Manipulation of fluids and reactions in microfluidic systems

ABSTRACT

Microfluidic structures and methods for manipulating fluids and reactions are provided. Such structures and methods may involve positioning fluid samples, e.g., in the form of droplets, in a carrier fluid (e.g., an oil, which may be immiscible with the fluid sample) in predetermined regions in a microfluidic network. In some embodiments, positioning of the droplets can take place in the order in which they are introduced into the microfluidic network (e.g., sequentially) without significant physical contact between the droplets. Because of the little or no contact between the droplets, there may be little or no coalescence between the droplets. Accordingly, in some such embodiments, surfactants are not required in either the fluid sample or the carrier fluid to prevent coalescence of the droplets. Structures and methods described herein also enable droplets to be removed sequentially from the predetermined regions.

FIELD OF INVENTION

The present invention relates generally to microfluidic structures, andmore specifically, to microfluidic structures and methods includingmicroreactors for manipulating fluids and reactions.

BACKGROUND

Microfluidic systems typically involve control of fluid flow through oneor more microchannels. One class of systems includes microfluidic“chips” that include very small fluid channels and smallreaction/analysis chambers. These systems can be used for analyzing verysmall amounts of samples and reagents and can control liquid and gassamples on a small scale. Microfluidic chips have found use in bothresearch and production, and are currently used for applications such asgenetic analysis, chemical diagnostics, drug screening, andenvironmental monitoring. Although these systems may allow manipulationof small volumes of fluids, additional methods that allow furthercontrol and flexibility are needed.

SUMMARY OF THE INVENTION

Microfluidic structures including microreactors for manipulating fluidsand reactions and methods associated therewith are provided.

In one aspect of the invention, a method is provided. The methodcomprises providing a microfluidic network comprising a first region anda microfluidic channel in fluid communication with the first region,flowing a first fluid in the microfluidic channel, flowing a firstdroplet comprising a second fluid in the microfluidic channel, whereinthe first fluid and the second fluid are immiscible, positioning thefirst droplet at the first region, and maintaining the first droplet atthe first region while the first fluid is flowing in the microfluidicchannel, wherein positioning and/or maintaining the first droplet at thefirst region does not require the use of a surfactant in the first orsecond fluids.

In some instances in connection with the methods described herein, thefirst and/or second fluids does not comprise a surfactant. The methodmay further comprise flowing a second droplet comprising a third fluidin the microfluidic channel, wherein the third fluid and the first fluidare immiscible, and positioning the second droplet at a second region influid communication with the microfluidic channel. In some instances,the third fluid does not comprise a surfactant. In other instances, thefirst and second droplets do not come into physical contact with eachother during the positioning and/or maintaining steps.

In some embodiments in connection with the methods described herein,positioning and/or maintaining the first droplet at the first region isindependent of flow rate of the first fluid in the microfluidic channel.

In some embodiments in connection with the methods described herein, thefirst region is closer in distance to a first inlet of the microfluidicnetwork for introducing the first fluid into the microfluidic channelthan the second region.

In some embodiments in connection with the methods described herein, thefirst droplet is positioned at the first region before the seconddroplet is positioned in the second region.

In some embodiments in connection with the methods described herein, themethod further comprises removing the first droplet from the firstregion and then removing the second droplet from the second region.

In some embodiments in connection with the methods described herein, themethod comprises flowing a fluid comprising a surfactant in themicrofluidic channel.

In some embodiments in connection with the methods described herein, themethod comprises coating the first and/or second droplets with asurfactant.

In some embodiments in connection with the methods described herein, themethod comprises dewetting the first and/or second droplets from asurface of the microfluidic channel.

In some embodiments in connection with the methods described herein, thefirst and second droplets are removed from the first and second regions,respectively, by reversing a direction of flow in the microfluidicchannel.

In some embodiments in connection with the methods described herein,positioning of the first droplet at the first region affects a directionof flow of a second droplet in the microfluidic network compared to whenthe first droplet is not positioned at the first region.

In some embodiments in connection with the methods described herein, thefirst droplet is positioned at the first region while the first fluid isflowing in the microfluidic channel.

In some embodiments in connection with the methods described herein, themicrofluidic channel comprises an upstream portion, a downstreamportion, and first and second fluid paths, at least one fluid pathbranching from the upstream portion and reconnecting at the downstreamportion.

In some embodiments in connection with the methods described herein, thefirst and second fluid paths have different resistances to flow.

In some embodiments in connection with the methods described herein, thefirst region is positioned within the first fluid path. In some cases,the first fluid path has less resistance to flow compared to the secondfluid path prior to positioning of a first droplet at the first region,and the first fluid path has greater resistance to flow afterpositioning of the first droplet at the first region.

In some embodiments in connection with the methods described herein, themethod comprises positioning several droplets at regions of themicrofluidic network, wherein the droplets are positioned in the regionsin the order the droplets are introduced into the microfluidic network.

In some embodiments in connection with the methods described herein, themethod comprises removing several droplets positioned at regions of themicrofluidic network, wherein the droplets are removed in the order thedroplets were introduced into the microfluidic network.

In some embodiments in connection with the methods described herein, themethod comprises removing several droplets positioned at regions of themicrofluidic network, wherein the droplets are removed in the reverseorder the droplets were introduced into the microfluidic network.

In another aspect of the invention, a method is provided. The methodcomprises providing a microfluidic network comprising at least a firstinlet tô a microfluidic channel, a first and a second region forpositioning a first and a′second droplet, respectively, the first andsecond regions in fluid communication with the microfluidic channel,wherein the first region is closer in distance to the first inlet thanthe second region, flowing a first fluid in the microfluidic channel,flowing a first droplet, defined by a fluid immiscible with the firstfluid, in the microfluidic channel, positioning the first droplet at thefirst region, flowing a second droplet, defined by a fluid immisciblewith the first fluid, in the microfluidic channel past the first regionwithout the second droplet physically contacting the first droplet, andpositioning the second droplet at the second region.

In one aspect of the invention, a method is provided. The methodcomprises positioning a first droplet defined by a first fluid, and afirst component within the first droplet, in a first region of amicrofluidic network, forming a first precipitate of the first componentin the first droplet while the first droplet is positioned in the firstregion, dissolving a portion of the first precipitate of the firstcompound within the first droplet while the first droplet is positionedin the first region, and re-growing the first precipitate of the firstcomponent in the first droplet.

In another aspect of the invention, a method is provided. The methodcomprises positioning a droplet defined by a first fluid, and a firstcomponent within the droplet, in a first region of a microfluidicnetwork, the droplet being surrounded by a second fluid immiscible withthe first fluid, positioning a third fluid in a reservoir positionedadjacent to the first region, the reservoir being separated from theregion by a semi-permeable barrier, changing a concentration of thefirst component within the first fluid of the droplet, and allowing aconcentration-dependent chemical process involving the first componentto occur within the droplet.

In another aspect of the invention, a method is provided. The methodcomprises positioning a droplet defined by a first fluid, and a firstcomponent within the droplet, in a first region of a microfluidicnetwork, the droplet being surrounded by a second fluid immiscible withthe first fluid, flowing a third fluid in a microfluidic channel influid communication with the first region and causing a portion of thesecond fluid to be removed from the first region, changing the volume ofthe droplet and thereby changing a concentration of the first componentwithin the droplet, and allowing a concentration-dependent chemicalprocess involving the first component to occur within the droplet.

In another aspect of the invention, a device is provided. The devicecomprises a fluidic network comprising a first region and a firstmicrofluidic channel allowing fluidic access to the first region, thefirst region constructed and arranged to allow a concentration-dependentchemical process to occur within said first region, wherein the firstregion and the first microfluidic channel are defined by voids within afirst material, a reservoir adjacent to the first region and a secondmicrofluidic channel allowing fluidic access to the reservoir, thereservoir defined at least in part by a second material that can be thesame or different than the first material, a semi-permeable barrierpositioned between the reservoir and the first region, wherein thebarrier allows passage of a first set of low molecular weight species,but inhibits passage of a second set of large molecular weight speciesbetween the first region and the reservoir, the barrier not constructedand arranged to be operatively opened and closed to permit and inhibit,respectively, fluid flow in the first region or the reservoir, whereinthe device is constructed and arranged to allow fluid to flow adjacentto a first side of the barrier without the need for fluid to flowthrough the barrier, and wherein the barrier comprises the firstmaterial, the second material, or a combination of the first and secondmaterials.

In another aspect of the invention, a method is provided. The methodcomprises providing a fluidic network comprising a first region, amicrofluidic channel allowing fluidic access to the first region, areservoir adjacent to the first region, and a semipermeable barrierpositioned between the first region and the reservoir, wherein the firstregion is constructed and arranged to allow a concentration-dependentchemical process to occur within the first region, and wherein thebarrier allows passage of a first set of low molecular weight species,but inhibits passage of a second set of large molecular weight speciesbetween the first region and the reservoir, providing a droplet definedby a first fluid in the first region, providing a second fluid in thereservoir, causing a component to pass across the barrier, therebycausing a change in a concentration of the first component in the firstregion, and allowing a concentration-dependent chemical processinvolving the first component to occur within the first region.

In another aspect of the invention, a method is provided. The methodcomprises providing a fluidic network comprising a first region and afirst microfluidic channel allowing fluidic access to the first region,the first region constructed and arranged to allow aconcentration-dependent chemical process to occur within said firstregion, wherein the first region and the microfluidic channel aredefined by voids within a first material, positioning a first fluidcontaining a first component in the first region, positioning a secondfluid in a reservoir via a second microfluidic channel allowing fluidicaccess to the reservoir, the reservoir and the second microfluidicchannel being defined by voids in a second material, and the reservoirbeing separated from the first region by a semi-permeable barrier,wherein the barrier comprises the first and/or second materials,changing a concentration of the first component in the first region, andallowing a concentration-dependent chemical process involving the firstcomponent to occur within the first region.

In another aspect of the invention, a method is provided. The methodcomprises positioning a first droplet defined by a first fluid, and afirst component within the droplet, in a first region of a microfluidicnetwork, positioning a second droplet defined by a second fluid, and asecond component within the droplet, in a second region of themicrofluidic network, wherein the first and second droplets are in fluidcommunication with each other, forming a first precipitate of the firstcomponent in the first droplet while the first droplet is positioned inthe first region, forming a second precipitate of the second componentin the second droplet while the second droplet is positioned in thesecond region, simultaneously dissolving a portion of the firstprecipitate and a portion of the second precipitate within the first andsecond droplets, respectively, and re-growing the first precipitate inthe first droplet and re-growing the second precipitate in the seconddroplet, while the first and second droplets are positioned in the firstand second regions, respectively.

In another aspect of the invention, a method is provided. The methodcomprises providing a microfluidic network comprising a first region anda microfluidic channel in fluid communication with the first region, thefirst region having at least one dimension larger than a dimension ofthe microfluidic channel, flowing a first fluid in the microfluidicchannel, flowing a first droplet comprising a second fluid in themicrofluidic channel, wherein the first fluid and the second fluid areimmiscible, and while the first fluid is flowing in the microfluidicchannel, positioning the first droplet in the first region, the firstdroplet having a lower surface free energy when positioned in the firstregion than when positioned in the microfluidic channel.

In another aspect of the invention, a method is provided. The methodcomprises providing a microfluidic network comprising a first region anda microfluidic channel in fluid communication with the first region,flowing a first fluid in the microfluidic channel, flowing a firstdroplet comprising a second fluid in the microfluidic channel, whereinthe first fluid and the second fluid are immiscible, while the firstfluid is flowing in the microfluidic channel, positioning the firstdroplet in the first region, and maintaining the first droplet in thefirst region while the first fluid is flowing in the microfluidicchannel.

In another aspect of the invention, a method is provided. The methodcomprises providing a microfluidic network comprising at least a firstinlet to a microfluidic channel, a first and a second region forpositioning a first and a second droplet, respectively, the first andsecond regions in fluid communication with the microfluidic channel,wherein the first region is closer in distance to the first inlet thanthe second region, flowing a first fluid in the microfluidic channel,flowing a first droplet, defined by a fluid immiscible with the firstfluid, in the microfluidic channel, while the first fluid is flowing inthe microfluidic channel, positioning the first droplet in the firstregion, flowing a second droplet, defined by a fluid immiscible with thefirst fluid, in the microfluidic channel, while the first fluid isflowing in the microfluidic channel, positioning the second droplet inthe second region, and maintaining the first droplet in the first regionand the second droplet in the second region, respectively, while thefirst fluid is flowing in the microfluidic channel.

In another aspect of the invention, a method is provided. The methodcomprises providing a microfluidic network comprising at least a firstinlet to a microfluidic channel, and a first and a second region forpositioning a first and a second droplet, respectively, the first andsecond regions in fluid communication with the microfluidic channel,flowing a first fluid at a first flow rate in the microfluidic channel,flowing a first droplet, defined by a fluid immiscible with the firstfluid, in the microfluidic channel, flowing a second droplet, defined bya fluid immiscible with the first fluid, in the microfluidic channel,flowing the first fluid at a second flow rate in the microfluidicchannel, wherein the second flow rate is slower than the first flowrate, and while the first fluid is flowing at the second flow rate,positioning the first droplet in the first region and positioning thesecond droplet in the second region.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIGS. 1A-1E show schematically a microfluidic network for positioning adroplet in a region of the network according to one embodiment of theinvention.

FIGS. 2A-2C show schematically removal of a droplet from a region of thenetwork according to another embodiment of the invention.

FIG. 3 is a photograph showing multiple sections of a microfluidicnetwork for positioning droplets according to another embodiment of theinvention.

FIG. 4 is a photograph showing multiple droplets positioned in multipleregions of a microfluidic network according to another embodiment of theinvention.

FIGS. 5A-5C show manipulation of a droplet positioned in a region of amicrofluidic network by changing the surface tension of the dropletaccording to another embodiment of the invention.

FIG. 6 is a photograph of a droplet wetting a surface of themicrofluidic network according to another embodiment of the invention.

FIG. 7 shows de-wetting of the droplet from a surface of themicrofluidic network after being treated with a stabilizing agentaccording to another embodiment of the invention.

FIGS. 8A-8D show schematically a microfluidic device for manipulatingfluids and reactions, according to another embodiment of the invention.

FIG. 9 shows schematically another microfluidic device for manipulatingfluids and reactions, according to another embodiment of the invention.

FIG. 10A is a photograph showing the formation of droplets, according toanother embodiment of the invention.

FIG. 10B shows a plot illustrating the combinatorial mixing of solutesin different droplets, according to another embodiment of the invention.

FIGS. 11A, 11A-1, 11B, 11B-1, 11C, 11D, 11E, 11E-1, 11F, and 11F-1 showthe positioning of droplets within micro wells of a microfluidic device,according to another embodiment of the invention.

FIGS. 12A-12B show the positioning of droplets within micro wells of amicrofluidic device using valves to open and close the entrance andexits of micro wells, according to another embodiment of the invention.

FIGS. 13A-13D show examples of changing the sizes of droplets in amicroreactor region of a device, according to another embodiment of theinvention.

FIGS. 14A-14G illustrate the processes of nucleation and growth ofcrystals inside a micro well of a device, according to anotherembodiment of the invention.

FIGS. 15A-15C show the increase and decrease of the size of a crystalinside a micro well of a device, according to another embodiment of theinvention.

FIG. 16A is a plot showing the relationship between free energy andcrystal nucleus size, according to another embodiment of the invention.

FIG. 16B is a phase diagram showing the relationship betweenprecipitation concentration and protein concentration, according toanother embodiment of the invention.

FIG. 17 is another phase diagram showing the relationship betweenprecipitation concentration and protein concentration, according toanother embodiment of the invention.

FIGS. 18A-18G show the use of another microfluidic device formanipulating fluids and reactions, according to another embodiment ofthe invention.

DETAILED DESCRIPTION

The present invention relates to microfluidic structures and methods formanipulating fluids and reactions. Such structures and methods mayinvolve positioning fluid samples, e.g., in the form of droplets, in acarrier fluid (e.g., an oil, which may be immiscible with the fluidsample) in predetermined regions in a microfluidic network, hi someembodiments, positioning of the droplets can take place in the order inwhich they are introduced into the microfluidic network (e.g.,sequentially) without significant physical contact between the droplets.Because of the little or no contact between the droplets, coalescencebetween the droplets can be avoided. Accordingly, in such embodiments,surfactants are not required in either the fluid sample or the carrierfluid to prevent coalescence of the droplets. Positioning of dropletswithout the use of surfactants is desirable in certain cases wheresurfactants may negatively interfere with the contents in the fluidsample (e.g., proteins). Structures and methods described herein alsoenable droplets to be removed sequentially from the predeterminedregions to a different region of the fluidic network where they can befurther processed.

Once the droplets are positioned at the predetermined regions, they canbe stored and/or may undergo manipulation (e.g., diffusion, evaporation,dilution, and precipitation). In some instances, many (e.g., 1000)droplets can be manipulated, sometimes simultaneously. Manipulation offluid samples can be useful for a variety of applications, includingtesting for reaction conditions, e.g., in crystallization, and chemicaland/or biological assays.

Microfluidic chips described herein may include a microfluidic networkhaving a region for forming droplets of sample in a carrier fluid (e.g.,an oil), and one or more microreactor regions (e.g., microwells,reservoirs, or portions of a microfluidic channel) in which the dropletscan be positioned and reaction conditions within the droplet can bevaried. Droplets may be positioned sequentially in regions of themicrofluidic network so that upon manipulating and/or performing achemical and/or biological process within each the droplets, thedroplets can be identified at a later time, for example, to determinethe particular conditions within the droplets that lead to a favorableoutcome (e.g., optimal conditions for forming a product, for crystalgrowth, etc.).

FIG. 1 shows a method for positioning a droplet in a region of amicrofluidic network according to one embodiment of the invention. Asshown in illustrative embodiment of FIG. 1A, microfluidic network 1000comprises section 1001 including microfluidic channel 1002 having anupstream portion 1006, a downstream portion 1010 (as fluid flows in thedirection of arrow 1012), and fluid paths 1014 and 1018. Fluid paths1014 and 1018 are connected, with fluid path 1018 branching off from theupstream portion and reconnecting at the downstream portion. Fluid paths1014 and 1018 may serve as alternative paths for fluid flow. In somecases, resistance to fluid flow may differ between fluid paths 1014 and1018. For example, fluid path 1014 may have less resistance to fluidflowing in the direction of arrow 1012 prior to positioning of a dropletin this section of the microfluidic network. As shown in thisillustrative embodiment, fluid path 1014 has a lower resistance to fluidflow than fluid path 1018 due to the relatively longer channel length offluid path 1018. It should be understood, however, that the microfluidicnetwork may have other designs and/or configurations for impartingdifferent relative resistances to fluid flow, and such designs andconfigurations can be determined by those of ordinary skill in the art.For instance, in some embodiments, the length, width, height, and/orshape of the fluid path can be designed to cause one fluid path to havea resistance to fluid flow different from another fluid path. In otherembodiments, at least a portion of a fluid path may include anobstruction such as a valve (which may change resistance dynamically), asemi-permeable plug (e.g., a hydrogel), a membrane, or another structurethat can impart and/or change resistance to fluid flow through thatportion.

As shown in FIGS. 1B and 1C, droplet 1020 flows in the direction of1012, e.g., by being carried by a carrier fluid flowing in the samedirection. Upon passing the junction between flow paths 1014 and 1018 atupstream portion 1006, the droplet flows in fluid path 1014 due to itslower resistance to flow in that fluid path relative to fluid path 1018.However, as fluid path 1014 includes a fluid constriction, e.g., in theform of a narrow fluid path portion 1024, droplet 1020 cannot flowfurther down the microfluidic network. Accordingly, droplet 1020 ispositioned within a region 1028 (e.g., a “micro well”) of themicrofluidic network. In some embodiments, droplet 1020 can bemaintained at the region even though carrier fluid continues to flow inthe microfluidic network (e.g., in the direction of arrow 1012).

Although FIG. 1 shows region 1028 having a cross-sectional areaapproximately the same as the cross-sectional area of microfluidicchannel 1002, it should be understood that region 1028 can have anysuitable cross-sectional area, dimensions, shape, etc. which may besuitable for containing, holding, and/or positioning a droplet. As shownin the embodiment illustrated in FIG. 1D, the positioning of droplet1020 at region 1028 causes fluid path 1014 to be plugged such that no orminimal fluid flows past narrow fluid path portion 1024. This pluggingof fluid path 1014 causes a higher resistance to fluid flow in that pathcompared to that of fluid path 1018. As a result, when a second droplet1030 flows in the direction of arrow 1012, the second droplet bypassesflow path 1014 and enters flow path 1018, which now has a lowerresistance than that of fluid path 1014 (FIG. 1D). Accordingly, seconddroplet 1030 can bypass first droplet 1020 and can now be positioned ina second region within microfluidic network 1000.

It should be understood that when droplet 1020 is positioned at region1028, the droplet may plug all or a portion of fluid path 1014 and/ornarrow fluid path portion 1024. For instance, in some cases, the dropletplugs all of such fluid paths such that none of the fluid flowing inmicrofluidic channel 1002 passes through narrow fluid path portion 1024.In other embodiments, the droplet may plug only a portion of such fluidpaths such that some fluid passes through narrow fluid path portion 1024even though the droplet is positioned at region 1028. The amount offluid flowing past the droplet may depend on factors such as thedimensions of fluid path portions 1014 and/or 1024, the size of thedroplets, the flow rate, etc. As long as the droplet causes fluid path1014 to have a higher relative resistance to fluid flow than fluid path1018, a second droplet can bypass fluid path 1014 and enter fluid path1018.

As described above, fluid paths 1014 and 1018 may have differentresistances fluid flow depending on whether or not a droplet ispositioned at region 1028. In the absence of a droplet positioned atregion 1028, fluid path 1014 may be configured to have a lowerresistance to fluid flow than fluid path 1018. For example, greater than50%, greater than 60%, greater than 70%, greater than 80%, or greaterthan 90% of the fluid flowing in channel 1002 at upstream portion 1006may flow in fluid path 1014 compared to fluid path 1018. However, whenthe droplet is positioned and maintained in region 1028, fluid path 1014may be relatively more restrictive to fluid flow. For example, less than50%, less than 40%, less than 30%, less than 20%, or less than 10% ofthe fluid flowing in channel 1002 at upstream portion 1006 may flow influid path 1014 compared to that of 1018. In some cases, 100% of thefluid flowing in direction 1012 in microfluidic channel 1002 flows influid path 1018 upon positioning of a droplet in region 1028.

As illustrated in the exemplary embodiment of FIG. 1, the positioning ofdroplet 1020 (e.g., a first droplet) and the subsequent bypass ofdroplet 1030 (e.g., a second droplet) does not require contact betweenthe first and second droplets. In certain embodiments, the seconddroplet does not physically contact the first droplet after positioningof the first droplet in region 1028. In other embodiments, the seconddroplet can come into physical contact with the first droplet as itbypasses the first droplet, however, due to such minimal contact betweenthe two droplets, the droplets do not coalesce. Accordingly, thepositioning of the droplets in the microfluidic network can take placewithout the use of surfactants. In other words, surfactants in either afluid flowing in channel 1002 (e.g., a carrier fluid) or within thedroplets is not required in order to stabilize the droplets and/orprevent the droplets from coalescing with one another during positioningor carrying the droplet in the microfluidic channel, and/or duringmaintaining the droplets within a predetermined region within themicrofluidic network. However, in instances where coalescence is desired(e.g., to allow a reaction between reagents contained in two droplets),the microfluidic network and methods for operating the network can beconfigured to allow such physical contact and/or coalescence betweendroplets.

In some embodiments, methods for positioning a droplet in a microfluidicnetwork include the steps of providing a microfluidic network comprisinga first region (e.g., region 1028 of FIG. 1A) and a microfluidic channelin fluid communication with the first region, flowing a first fluid(e.g., a carrier fluid) in the microfluidic channel, and flowing a firstdroplet comprising a second fluid (e.g., a fluid sample) in themicrofluidic channel, wherein the first fluid and the second fluid areimmiscible. The first droplet may be positioned in the first region andmaintained in the first region while the first fluid is flowing in themicrofluidic channel. In such embodiments, positioning and/ormaintaining the first droplet in the first region does not require theuse of a surfactant in the first or second fluids.

In some embodiments, a chemical and/or biological process can be carriedout in droplet 1020 of FIG. 1 while the droplet is positioned in region1028. Additionally or alternatively, the droplet may be manipulated. Forexample, a fluid sample in the droplet may undergo a process such asdiffusion, evaporation, dilution, and/or precipitation. Such methods ofmanipulation are described in more detail below. The droplet may bemanipulation by, for example, changing the concentration of the fluidflowing in channel 1002 after the droplet has been positioned at region1028. In other embodiments, region 1028 is in fluid communication withanother fluidic channel, flow path, reservoir, or other structure, e.g.,via a semi permeable membrane that may be positioned adjacent the region(e.g., underneath or above region 1028), and manipulation of the dropletcan occur via such passages.

In some embodiments of the invention, droplets that have been positionedat regions of a microfluidic network can be removed or extracted fromthe regions to a different location in the fluidic network, where theycan be optionally processed, manipulated, and/or collected. As shown inthe illustrative embodiments of FIGS. 2A-2C, removing droplet 1020 fromregion 1028 of section 1001 of microfluidic network 1000 can take placeby reversing the flow of the carrier fluid in the network such that thecarrier fluid now flows in the direction of arrow 1040 (instead of inthe direction of arrow 1012 of FIGS. 1A-1E).

In such embodiments, upstream portion 1006 and downstream portion 1010of FIGS. 1A-1E now become reversed such that portion 1010 is now anupstream portion and portion 1006 is now a downstream portion. The flowof a carrier fluid in the direction of arrow 1040 in microfluidicchannel 1002 causes a portion of the fluid to flow through narrow fluidpath portion 1024 into region 1028 where droplet 1020 is positioned.This fluid flow causes the droplet to flow in the direction of arrow1040. As shown in the embodiment illustrated in FIG. 2B, droplet 1030,which may have been positioned at a different region of the microfluidicnetwork, can be removed from that region and may also flow in thedirection of arrow 1040. As droplet 1030 encounters narrow fluid pathportion 1024, the droplet cannot flow through this narrow opening due toits high resistance to flow. As a result, the droplet bypasses thenarrow fluid path portion and flows into fluid path 1018 until itreaches microfluidic channel 1002 at downstream portion 1006. Thus, byreversing the flow and the pressure gradient in the microfluidicnetwork, droplets 1020 and 1030 can be removed sequentially from theregions of the microfluidic network where they previously resided. Thatis, droplet 1020, which was positioned first before droplet 1030, can beremoved from its region and can enter a different region of themicrofluidic network before that of droplet 1020.

In some embodiments, sequential positioning of droplets can be performedsuch that a first droplet is positioned in a first region before asecond droplet is positioned in a second region (and, optionally, beforethird, fourth, fifth droplets, etc. are positioned in their respectiveregions). As described above, sequential removal of the droplets can beperformed such that the first droplet is removed from a region and/orpositioned at a different location on the microfluidic network beforethe second droplet (and, optionally, before third, fourth, fifthdroplets, etc. are removed from their respective regions). In otherembodiments, removal of the droplets can be performed such that thesecond droplet is removed and/or positioned at a different location onthe microfluidic network before the first droplet.

In some cases, several (e.g., greater than 2, greater than 5, greaterthan 10, greater than 50, greater than 100, greater than 200, greaterthan 500, or greater than 1000) droplets can be positioned at regions ofthe microfluidic network, wherein the droplets are positioned in theregions in the order the droplets are introduced into the microfluidicnetwork. In some cases, removing several droplets positioned at regionsof the microfluidic network comprises removing the droplets in the orderthe droplets were introduced into the microfluidic network (or in theorder the droplets were positioned into the regions of the microfluidicnetwork). In other cases, removing several droplets positioned atregions of the microfluidic network comprises removing the droplets inthe reverse order the droplets were introduced into the microfluidicnetwork (or in the reverse order the droplets were positioned into theregions of the microfluidic network). Other methods of positioning andremoval of droplets are also possible.

The sequential (or predetermined/known order of) removal of dropletsfrom regions of a microfluidic network can allow control over theidentification and location of each droplet within the network. This canalso allow determination of the contents inside each of the dropletsfrom the time they are formed and/or introduced into the microfluidicnetwork, to the time the droplets are manipulated and/or extracted fromthe microfluidic network.

FIG. 3 is a photograph of multiple sections 1001-A, 1001-B, and 1001-Cof microfluidic network 1050 according to one embodiment of theinvention. A carrier fluid may flow in microfluidic channel 1002-A inthe direction of arrow 1012 from an inlet positioned upstream fromportion 1006. The carrier fluid may partition at the junction wherefluid paths 1014-A and 1018-A branch off from microfluidic channel 1002.The proportion of fluid that flows in each of the fluid paths can bedetermined at least in part by the relative resistance to fluid flow inthe paths, as described above. In the embodiment shown in FIG. 3,sections 1001-A, 1001-B, and 1001-C are positioned in series. In otherembodiments, however, such sections may be positioned in parallel and/orin both series and parallel. Other configurations are also possible.

A microfluidic network may have any suitable number of microfluidicsections 1001. For instance, the microfluidic network may have greaterthan or equal to 5, greater than or equal to 10, greater than or equalto 30, greater than or equal to 70, greater than or equal to 100,greater than or equal to 200, greater than or equal to 500, or greaterthan or equal to 1000 such sections.

In additional, although certain embodiments herein show that sections1001 can allow positioning of a single droplet in each of the sections,in other embodiments, the sections can be designed such that greaterthan one droplet (e.g., greater than or equal to 2, greater than orequal to 5, or greater than or equal to 10 droplets) can be positionedat each section.

Furthermore, although only two fluid flow paths 1014 and 1018 are shownbranching off from channel 1002, in other embodiments, more than two(e.g., greater than or equal to 3, greater than or equal to 5, orgreater than or equal to 10) fluid paths may branch off from channel1002. Each branching fluid path may optionally comprise one or moreregions (e.g., microwells) for positioning and/or maintaining droplets.

FIG. 4 shows the positioning of droplets 1060, 1062, and 1064 atpositions 1028-A, 1028-B, and 1028-C, respectively, in micro fluidicnetwork 1050 according to one embodiment of the invention. As shown inthis illustrative embodiment, carrier fluid flows in the direction ofarrow 1012 and carries droplet 1060 through channel 1002-A and intofluidic path 1014-A due to the lower resistance to fluid flow in thatfluid path compared to that of fluid path 1018-A. That is, prior to thepositioning of droplet 1060 in region 1028-A, more than 50% of the fluidflowing in microfluidic channel 1002-A flows through fluid path 1014-Acompared to fluid path 1018-A.

Once droplet 1060 is positioned at region 1028-A, it impedes fluid flowthrough narrow fluid path portion 1024-A such that the resistance tofluid flow in fluid paths 1014-A and 1018-A are altered. This causesresistance to fluid flow to be higher in portion 1014-A and as a result,a greater amount of fluid flows in the direction of 1070 through fluidpath portion 1018-A. Accordingly, a second droplet 1062 flowing throughmicrofluidic channel 1002-A and passing upstream portion 1006 nowbypasses fluid path portion 1014-A and flows through portion 1018-A. Thesecond droplet, after bypassing region 1028-A, now enters microfluidicchannel portion 1002-B. If there is a lower resistance to fluid flow influid path portion 1014-B (e.g., a droplet has not already beenpositioned in region 1028-B), the droplet can be positioned at thisregion. Next, a third droplet 1064 can flow through microfluidic channelportion 1002-A in the direction of arrow 1012 and first bypasses region1028-A due to droplet 1060 already positioned at that region. Thedroplet can then flow into fluid path portion 1018-A and 1002-B. Sincedroplet 1062 has already been positioned at region 1028-B, third droplet1064 bypasses this region and takes the fluid path of least resistance(fluid path portion 1018-B). Upon entering an empty region such as1028-C, the third droplet can now position itself at that region due toa lower resistance to fluid flow in fluid path 1014-C compared to thatof fluid path portion 1018-C (e.g., prior to any other droplet beingpositioned at region 1028-C).

Accordingly, a method for positioning droplets in regions of amicrofluidic network may include providing a microfluidic networkcomprising at least a first inlet to a microfluidic channel (e.g.,positioned upstream of portion 1006 of FIG. 4), a first region (e.g.,region 1028-A) and a second region (e.g., region 1028-B) for positioninga first and a second droplet, respectively, the first and second regionsin fluid communication with the microfluidic channel, wherein the firstregion is closer in distance to the first inlet than the second region.The method can include flowing a first fluid (e.g., a carrier fluid) inthe microfluidic channel, flowing a first droplet (e.g., a first fluidsample), defined by a fluid immiscible with the first fluid, in themicrofluidic channel, and positioning the first droplet in the firstregion. The method can also include flowing a second droplet (e.g., asecond fluid sample), defined by a fluid immiscible with the firstfluid, in the microfluidic channel past the first region without thesecond droplet physically contacting the first droplet. The seconddroplet may be positioned at the second region. In some instances, thefirst and/or second droplets are maintained at their respective regionswhile fluid continues to flow in the microfluidic channel.

It should be understood that other components may be integrated withfluidic networks described herein in some embodiments of the invention.For example, in some instances, resistances to fluid flow can be changeddynamically such that the direction of fluid flow (and, therefore,positioning of droplets) can be controlled by the user. In one suchembodiment, valves may be positioned at one or more of positions 1070-A,1070-B, and 1070-C of FIG. 4. For example, a valve at position 1070-Bcan cause restriction of fluid flow through fluid path portion 1014-B,e.g., prior to a droplet being positioned at region 1028-B. This cancause a droplet flowing through microfluidic channel portion 1002-B tobypass region 1028-B even though a droplet is not positioned at thatregion. Thus, the droplet flowing through portion 1002-B will flowthrough fluid path 1018-B and onto the next available region, where thefluid resistance of that region may or may not be controlled by asimilar valve. In some instances, after a droplet bypasses region 1028-Bdue to a closed valve at position 1070-B (or any other component thatcan change the relative resistances to fluid flow between fluid paths1014-B and 1018-B), the valve at position 1070-B can now be reopened tochange the relative resistances to fluid flow such that a next dropletcan now enter into region 1028-B and be positioned at that region. Sucha system can allow droplets to be positioned at any desired region of amicrofluidic network.

As described herein, in some embodiments droplets do not requirestabilization (e.g., the use of surfactants or other stabilizing agents)in order to be positioned at predetermined regions within microfluidicnetworks described herein. This is because in some embodiments, thedroplets do not significantly physically contact one another duringbypass of one droplet to another. Due to the little or no physicalcontact between the droplets, the droplets do not have a chance tocoalesce with one another. Thus, surfactants or other stabilizing agentsare not required to stabilize the droplets from coalescence in suchembodiments. In some embodiments, the absence of surfactants or otherstabilizing agents causes the droplets to wet a surface of themicrofluidic network. Even though wetting may occur, the droplets canstill be positioned at predetermined regions within the microfluidicnetwork due to, for example, a positive pressure that causes fluid flowto carry these droplets into these regions. As discussed above, the useof droplets and/or a carrier fluid that does not contain a surfactant isadvantageous in some embodiments where surfactants may negativelyinterfere with contents inside the droplets. For example, the dropletsmay contain proteins, and surfactants are known to denature certainproteins to some extent. However, after manipulation of the dropletand/or carrying out a process such as a chemical and/or biologicalreaction inside the droplet, surfactants may no longer negatively affectthe contents inside the droplet. Accordingly, in such cases, asurfactant or other stabilizing agent can be applied to the droplets. Insome embodiments, such application of a stabilizing agent to a dropletafter manipulation of the droplet and/or carrying out a process insidethe droplet can facilitate mobilization of the droplet out of the regionin which the droplet is positioned.

It should be understood, however, than in some embodiments, a dropletand/or a carrier fluid may contain a surfactant or other stabilizingagent that stabilizes a droplet prior to positioning of the droplet at aregion in the microfluidic network. In such embodiments, thestabilization may not negatively interfere with contents (e.g.,reagents) inside the droplet. Of course, such embodiments will depend ona variety of factors such as the type of stabilizing agent used, thecontents inside the droplet, the application, etc.

FIGS. 5A-5C show schematically the treatment of a droplet positioned ata predetermined region within a microfluidic network with a stabilizingagent according to one embodiment of the invention. As described above,droplet 1080 can be positioned at region 1028 by flowing a carrier fluidand the droplet in the direction of arrow 1012. After the droplet hasbeen positioned, the droplet may wet a surface of the channel, such assurface portions 1084. In some embodiments, this can cause the dropletto be immobilized at this region, even when a carrier fluid is flowed inthe opposite direction (e.g., in the direction of arrow 1088) in attemptto remove the droplet from this region. In such embodiments, a fluidcomprising a stabilizing agent (e.g., a surfactant) can be flowed in thedirection of arrow 1088 through microfluidic channel 1002. A portion ofthis fluid can flow through narrow path portion 1024 to reach droplet1080 at region 1028. This fluid containing the stabilizing agent cancause the droplet to be coated with the stabilizing agent, which canresult in the droplet de-wetting from the channel at surface portions1084. In such cases, the surface tension of the droplet has beenreduced. Thus, the droplet may be “depinned” from one or more surfacesof the channel. If desired, after introduction of a fluid containing astabilizing agent to the droplet, the fluid flow may be stopped for acertain amount of time to allow the stabilizing agent to coat thedroplet. In other embodiments, however, flow in channel 1002 is notstopped after the stabilizing agent has been introduced. In yet otherembodiments, after a droplet has been de-wetted from a surface of themicrofluidic network, fluid flowing in the microfluidic network may bereplaced by a second fluid (which may or may not contain a stabilizingagent). As shown in the embodiment illustrated in FIG. 5C, droplet 1080can be removed/extracted from region 1028 in the direction of arrow1088. One of ordinary skill in the art can determine appropriateconditions for de-wetting a droplet from a surface of the microfluidicnetwork which may depend on conditions such as the concentration of thestabilizing agent in the fluid, the flow rate, the degree of wetting ofthe droplet, the contents of the droplet, the material composition ofthe fluidic network, as well as other factors.

FIG. 6 is a photograph showing droplet 1092 that has wetted surfaceportions 1096 of microfluidic network 1090 at region 1028. As shown inFIG. 7, after flowing a fluid containing a surfactant in the directionof arrow 1088, a portion of which flows through a narrow path portion1024, droplet 1092 de-wets surface portions 1096 and is now stabilizedwith the stabilizing agent. The stabilization is evident by meniscus1098 that forms around droplet 1092, as the droplet now takes on a lowerenergy state configuration compared to that shown in FIG. 6.

It should be understood that a fluid containing a stabilizing agent canbe introduced into microfluidic network 1090 in any suitable manner. Forexample, in some embodiments, the stabilizing agent may be introduced bya fluid flowing in the direction of arrow 1012. In other embodiments,region 1028 may be in fluidic communication with another portion of thedevice branching from region 1028. For instance, above or below region1028 may be a reservoir, a channel, or other component that can be usedto introduce a stabilizing agent or other entity to a droplet in thatregion.

As shown in FIGS. 2 and 5, droplets that are released from a region of amicrofluidic network can be caused to flow in a direction opposite thatwhich was used to position the droplet in the region. In otherembodiments, however, after a droplet has been removed from region inwhich it was positioned, the droplet may be caused to flow in the samedirection as that which was used to position the droplet. For example,in one embodiment, droplet 1080 of FIG. 5C can be released from position1028 and can be caused to flow in the direction of 1088 until thedroplet resides at a downstream portion of channel 1002 (e.g., at thetop of microfluidic network 1000 as shown in FIG. 5C). Then, a valve orother component that may be positioned at position 1070 can be at leastpartially closed to cause a higher resistance to fluid flow in fluidflow path 1014 compared to that of 1018. Since fluid flow path 1018 nowhas a lower resistance to fluid flow, flow of the carrier fluid can nowbe reversed such that it flows in the direction of arrow 1012, in whichcase the droplet can bypass fluid flow path 1014 and enter fluid flowpath 1018.

As described above, methods for storing and/or extracting droplets in amicrofluidic network are provided herein. In some embodiments, thedroplets may be stored and/or extracted in sequential order. Forexample, the droplets may be extracted in the order they are stored orpositioned in predetermined regions in the microfluidic network.Advantageously, in some embodiments, such methods do not require the useof surfactants or other stabilizing agents, since the droplets may notcome into substantial physical contact with one another in a manner thatcauses coalescence. This is advantageous in certain cases as surfactantsmay interfere with contents such as proteins inside the droplet, as isknown to those of ordinary skill in the art. In some embodiments, themicrofluidic networks shown in FIGS. 1-7 can be combined with one ormore of the features shown in FIGS. 8-18 below. For instance, regions1028 of FIG. 1A for positioning a droplet can replace microwells 2130 ofFIGS. 8 and 14 and/or micro wells or regions in other embodiments shownin FIGS. 8-18. Thus, the microfluidic networks of FIGS. 1-7 may be apart of the microfluidic chips described in connection with FIGS. 8-18and may be connected to any suitable component shown in these figures.

As described above, microfluidic chips described herein may include aregion for forming droplets of sample in a carrier fluid (e.g., an oil),and one or more microreactor regions (also called “predeterminedregions” or “regions” herein) in which the droplets can be positionedand reaction conditions within the droplet can be varied. For instance,one such system includes microreactor regions containing several (e.g.,1000) microwells or other structures that are fluidically connected to amicrochannel, or formed as a part of the microchannel. A reservoir(i.e., in the form of a chamber or a channel) for containing a gas or aliquid can be situated underneath a microwell, separating the microwellby a semi-permeable barrier (e.g., a dialysis membrane). In some cases,the semi-permeable barrier enables chemical communication of certaincomponents between the reservoir and the microwell; for instance, thesemi-permeable barrier may allow water, but not proteins, to pass acrossit. Using the barrier, a condition in the reservoir, such asconcentration or ionic strength, can be changed (e.g., by replacing thefluid in the reservoir), thus causing the indirect change in a conditionof a droplet positioned inside the microwell. This format allows controland the testing of many reaction conditions simultaneously. Microfluidicchips and methods of the invention can be used in a variety of settings.One such setting, described in more detail below, involves the use of amicrofluidic chip for crystallizing proteins within aqueous droplets offluid. Advantageously, crystallization conditions can be controlled suchthat nucleation and growth of crystals can be decoupled, performedreversibly, and controlled independently of each other, thereby enablingthe formation of defect-free crystals.

FIGS. 8A-8C illustrate a microfluidic chip 2010 according to oneembodiment of the invention. As shown in FIG. 8 A, microfluidic chip2010 contains a droplet formation region 2015 connected fluidically toseveral microreactor regions 2020, 2025, 2030, 2035, and 2040. Thedroplet formation region can include several inlets 2045, 2050, 2055,2060, and 2065, which may be used for introducing different fluids intothe chip. For instance, inlets 2050, 2055, and 2060 may each containdifferent aqueous solutions necessary for protein crystallization. Therate of introduction of each of the solutions into inlets 2050, 2055,and 2060 can be varied so that the chemical composition of each of thedroplets is different, as discussed in more detail below. Inlets 2045and 2065 may contain a carrier fluid, such as an oil immiscible with thefluids in inlets 2050, 2055, and 2060. Fluids in inlets 2050, 2055, and2060 can flow (i.e., laminarly) and merge at intersection 2070. Whenthis combined fluid reaches intersection 2075, droplets of aqueoussolution can be formed in the carrier fluid. Droplet formation region2015 also includes a mixing region 2080, where fluids within eachdroplet can mix, e.g., by diffusion or by the generation of chaoticflows.

Droplets formed from region 2015 can enter one, or more, of microreactorregions 2020, 2025, 2030, 2035, or 2040 via channel 2085. The particularmicroreactor region in which the droplets enter can be controlled byvalves 2090, 2095, 2100, 2105, 2110, and/or 2111, which can be activatedby valve controls 2092, 2094, 2096, 2098, 2102, 2104, 2106, 2108, 2112,2114, and/or 2116. For example, for droplets to enter microreactorregion 2020, valve 2090 can be opened by activating valve controls 2092and 2094, while valves 2095, 2100, 2105, 2110, and 2111 are closed. Thismay allow the droplets to flow into channel 2115 in the direction ofarrow 2120, and then into channel 2125 and to several microwells 2130(FIGS. 8B and 8C). As discussed in more detail below, each droplet canbe positioned in a microwell, i.e., by the use of surface tensionforces. Any of a number of valves and/or pumps, including peristalticvalves and/or pumps, suitable for use in a fluidic network such as thatdescribed herein can be selected by those of ordinary skill in the artincluding, but not limited to, those described in U.S. Pat. No.6,767,194, “Valves and Pumps for Microfluidic Systems and Methods forMaking Microfluidic Systems”, and U.S. Pat. No. 6,793,753, “Method ofMaking a Microfabricated Elastomeric Valve,” which are incorporatedherein by reference.

As shown in FIG. 8C, microwells 2130 (as well as channels 2115 and 2125,and other components) can be defined by voids within structure 2135,which can be made of a polymer such as poly(dimethylsiloxane) (PDMS).Structure 2135 can be supported by optional support layers 2136 and/or2137 which can be fully or partially polymeric or made of anothersubstance including ceramic, silicon, or other material selected forstructural rigidity suitable for the intended purpose of the particulardevice. As illustrated in this embodiment, reservoir 2140 and posts 2145are positioned below microwells 2130 as part of layer 2149, and separatethe microwells by a semi-permeable barrier 2150. In the embodimentillustrated in FIG. 8C, semi-permeable barrier is formed in layer 2149.In some instances, semi-permeable barrier 2150 allows certain lowmolecular weight components (e.g., water, vapor, gases, and lowmolecular weight organic solvents such as dioxane and iso-propanol) topass across it, while preventing larger molecular weight components(e.g., salts, proteins, and hydrocarbon-based polymers) and/or certainfluorinated components (e.g., fluorocarbon-based polymers) from passingbetween microwells 2130 and reservoir 2140. By controlling thesubstances entering reservoir inlet 2155 (i.e., for microreactor region2020), a condition (e.g., concentration, ionic strength, or type offluid) in the reservoir can be changed. This can result in the change ofa condition in microwells 2130 indirectly by a process such as diffusionand/or by flow of components past barrier 2150, as discussed below.Because there may be several (e.g., 1000) microwells on a chip, manyreaction conditions can be tested simultaneously. Once a reaction hasoccurred in a droplet, the droplet can be transported, e.g., out of thedevice or to another portion of the device, for instance, via outlet2180.

FIG. 8D shows an alternative configuration for the fabrication of device2010. As illustrated in this figure, layer 2149 comprising reservoir2140 is positioned above structure 2135 comprising microwells 2130 andchannel 2125. In this embodiment, semi-permeable barrier 2150 is formedas part of structure 2135 i.e., by spin coating.

In the embodiment illustrated in FIG. 8D the membrane is fabricated aspart of the layer containing the microwells, while as shown in FIG. 8C,the semi-permeable membrane is fabricated as part of the reservoirlayer. In each case the layer containing the membrane can be thin (e.g.,less than about 20 microns thick) and can be fabricated via spincoating, while the other layer(s) can be thick (e.g., greater than about1 mm) and may be fabricated by casting a fluid. In other embodiments,however, semi-permeable barrier can be formed independently of layers2149 and/or structure 2135, as described in more detail below.

It is to be understood that the structural arrangement illustrated inthe figures and described herein is but one example, and that otherstructural arrangement can be selected. For example, a microfluidicnetwork can be created by casting or spin coating a material, such as apolymer, from a mold such that the material defines a substrate having asurface into which are formed channels, and over which a layer ofmaterial is placed to define enclosed channels such as microfluidicchannels. In another arrangement a material can be cast, spin-coated, orotherwise formed including a series of voids extending throughout onedimension (e.g., the thickness) of the material and additional materiallayers are positioned on both sides of the first material, partially orfully enclosing the voids to define channels or other fluidic networkstructures. The particular fabrication method and structural arrangementis not critical to many embodiments of the invention. In other cases, aparticular structural arrangement or set of structural arrangements candefine one or more aspects of the invention, as described herein.

FIG. 9 shows another exemplary design of a microfluidic chip, device2000, which includes droplet formation region 2015, buffer region 2022,micro well region 2024, and microreactor region 2026. Buffer region 2022can be used, for example, to allow a droplet formed in the dropletregion to equilibrate with a carrier fluid. The buffer region isconnected to microwell region 2024, which can be used for storingdroplets. Microwell region 2024 is connected to microreactor region2026, which contains microwells and reservoir channels positionedbeneath the microwells, i.e., for changing a condition within dropletsthat are stored in the microwells. Droplets formed at intersection 2075can enter regions 2022, 2024, or 2026, depending on the actuation of aseries of valves. For instance, a droplet can enter buffer region 2022by opening valve 2090 and 2100, while closing valve 2091. A droplet canenter microwell region 2024 directly by opening valves 2090, 2091, 2093,and 2095 while closing valves 2097, 2099, 2100, and 2101.

The formation of droplets at intersection 2075 of device 2000 is shownin FIG. 10A. As shown in this diagram, fluid 2054 flows in channel 2056in the direction of arrow 2057. Fluid 2054 may be, for example, anaqueous solution containing a mixture of components from inlets 2050,2055, 2060, and 2062 (FIG. 9). Fluid 2044 flows in channel 2046 in thedirection of arrow 2047, and fluid 2064 flows in channel 2066 in thedirection of arrow 2067. In this particular embodiment, fluids 2044 and2064 have the same chemical composition and serve as a carrier fluid2048, which is immiscible with fluid 2054. In other embodiments,however, fluids 2044 and 2064 can have different chemical compositionsand/or miscibilities relative to each other and to fluid 2054. Atintersection 2075, droplets 2077, 2078, and 2079 are formed byhydrodynamic focusing after passing through nozzle 2076. These dropletsare carried (or flowed) in channel 2056 in the direction of arrow 2057.

Droplets of varying sizes and volumes may be generated within themicrofluidic system. These sizes and volumes can vary depending onfactors such as fluid viscosities, infusion rates, and nozzlesize/configuration. In some cases, it may be desirable for each dropletto have the same volume so that different conditions (e.g.,concentrations) can be tested between different droplets, while theinitial volumes of the droplets are constant. In other cases, it may besuitable to generate different volumes of droplets for use in an assay.Droplets may be chosen to have different volumes depending on theparticular application. For example, droplets can have volumes of lessthan 1 μL, less than 0.1 μL, less than 10 nL, less than 1 nL, less than0.1 nL, or less than 10 pL. It may be suitable to have small droplets(e.g., 10 pL or less), for instance, when testing many (e.g., 1000)droplets for different reaction conditions so that the total volume ofsample consumed is low. On the other hand, large (e.g., 10 nL-1 μL)droplets may be suitable, for instance, when a reaction condition isknown and the objective is to generate large amounts of product withinthe droplets.

The rate of droplet formation can be varied by changing the flow ratesof the aqueous and/or oil solutions (or other combination of immisciblefluids defining carrier fluid and droplet, which behave similarly to oiland water, and which can be selected by those of ordinary skill in theart). Any suitable flow rate for producing droplets can be used; forexample, flow rates of less than 100 nL/s, less than 10 nL/s, or lessthan 1 nL/s. In one embodiment, droplets having volumes between 0.1 to1.0 nL can be formed while flow rates are set at 100 nL/s. Under theseconditions, droplets can be produced at a frequency of 100 droplets/s.In another embodiment, the flow rates of two aqueous solutions can bevaried, while the flow rate of the oil solution is held constant, asdiscussed in more detail below.

FIG. 11 shows one example of a method for positioning droplets withinregions of a microfluidic channel. In the embodiment illustrated in FIG.11A, carrier fluid 2048 flows in channel 2056 in the direction of arrow2057 while droplets 2078 and 2079 are positioned in microwells 2082 and2083, respectively. Droplet 2077 is carried in fluid 2048 also in thedirection of arrow 2057. Droplet 2077 passes and may physically contactdroplet 2079, but does not coalesce with droplet 2079 since the surfacesof the droplets may include a surfactant that prevents coalescence. Asshown in FIG. 11B, when droplet 2077 is adjacent to microwell 2082,droplet 2077 tries to enter into this microwell. Since droplet 2078 hasalready occupied microwell 2082, however, droplet 2077 cannot fit anddoes not enter into this microwell. Meanwhile, the pressure of thecarrier fluid pushes droplet 2077 forward in the direction of arrow2057. When droplet 2077 passes an empty microwell, e.g., microwell 2081,droplet 2077 can enter and be positioned in this microwell (FIGS.11D-11F). In a similar manner, the next droplet behind (i.e., to theleft of) droplet 2077 can fill the next available microwell to the rightof microwell 2081 (not shown). The passing of one droplet over anotherthat has already been positioned into a microwell is referred to as the“leapfrog” method. In the leapfrog method, the most upstream microwellcan contain the first droplet formed and the most downstream microwellcan contain the last droplet formed.

Because droplets are carried past each other (e.g., as in FIGS.11A-11C), and/or for other reasons involving various embodiments of theinvention, a surfactant may be added to the droplet to stabilize thedroplets against coalescence. Any suitable surfactant such as adetergent for stabilizing droplets can be used, including anionic,non-ionic, or cationic surfactants. In one embodiment, a suitabledetergent is the non-ionic surfactant Span 80, which does not denatureproteins yet stabilizes the droplets. Criteria for choosing othersuitable surfactants are discussed in more detail below.

Different types of carrier fluids can be used to carry droplets in adevice. Carrier fluids can be hydrophilic (i.e., aqueous) or hydrophobic(i.e., an oil), and may be chosen depending on the type of droplet beingformed (i.e., aqueous or oil-based) and the type of process occurring inthe droplet (i.e., crystallization or a chemical reaction). In somecases, a carrier fluid may comprise a fluorocarbon. In some embodiments,the carrier fluid is immiscible with the fluid in the droplet. In otherembodiments, the carrier fluid is slightly miscible with the fluid inthe droplet. Sometimes, a hydrophobic carrier fluid, which is immisciblewith the aqueous fluid defining the droplet, is slightly water soluble.For example, oils such as PDMS and poly(trifluoropropylmethylsiloxane)are slightly water soluble. These carrier fluids may be suitable whenfluid communication between the droplet and another fluid (i.e., a fluidin the reservoir) is desired. Diffusion of water from a droplet, throughthe carrier fluid, and into a reservoir containing air is one example ofsuch a case.

A droplet can enter into an empty microwell by a variety of methods. Inthe embodiment shown in FIG. 11A, droplet 2077 is surrounded by an oiland is forced to flow through channel 2056, which has a large width(W₅₆), but small height (h₅₆). Because of its confinement, droplet 2077has an elongated shape while positioned in channel 2056, as the top,bottom, and side surfaces of the droplet take on the shape of thechannel. This elongated shape imparts a high surface energy on thedroplet (i.e., at the oil/water interface) compared to the same droplethaving a spherical shape (i.e., of the same volume). When droplet 2077passes an empty microwell 2081, which has a larger cross-sectionaldimension (e.g., height, h₁₃₀) than that of channel 2056, droplet 2077favors the microwell since the dimensions of the microwell allow thedroplet to form a more spherical shape (as shown in FIG. 11F), therebylowering its surface energy. In other words, when droplet 2077 isadjacent to empty microwell 2081, the gradient between the height of thechannel and the height in the microwell produces a gradient in thesurface area of the droplet, and therefore a gradient in the interfacialenergy of the droplet, which generates a force on the droplet driving itout of the confining channel and into the microwell. Using this method,droplets can be positioned serially in the next available microwell(e.g., an empty microwell) while the carrier fluid is flowing. In otherembodiments, methods such as patterned surface energy, electrowetting,and dielectrophoresis can drive droplets into precise locations inmicrofluidic systems.

In another embodiment, a method for positioning droplets into regions(e.g., microwells) of a microfluidic network comprises flowing aplurality (e.g., at least 2, at least 10, at least 50, at least 100, atleast 500, or at least 1,000) of droplets in a carrier fluid in amicrofluidic channel at a first flow rate. The first flow rate may befast, for instance, for forming many droplets quickly and/or for fillingthe microfluidic network quickly with many droplets. At a fast flowrate, the droplets may not position into the regions. When the carrierfluid is flowed at a second flow rate slower than the first flow rate,however, each droplet may position into a region closest to the dropletand remain in the region. This method of filling microwells is referredto as the “fast flow/slow flow” method. Using this method, the dropletscan be positioned in the order that the droplets are flowed into thechannel, although in some instances, not every region may be filled(i.e., a first and a second droplet that are positioned in theirrespective regions may be separated by an empty region). Since thismethod does not require droplets to pass over filled regions (e.g.,microwells containing droplets), as is the case as shown in FIG. 11, thedroplets may not require surfactants when this method of positioning isimplemented.

Another method for filling microwells in the order that the droplets areformed is by using valves at entrances and exits of the microwells, asshown in FIG. 12. In this illustrative embodiment, droplets 2252, 2254,2256, 2258, 2260, and 2262 are flowed into device 2250 comprisingchannels 2270, 2271, 2272, and 2273, and microwells 2275, 2280, 2285,and 2290. Each microwell can have an entrance valve (e.g., valves 2274,2279, 2284, and 2289) and an exit valve (e.g., valves 2276, 2281, 2286,and 2291) in either opened or closed positions. For illustrativepurposes, opened valves are marked as “o” and closed valves are markedas “x” in FIG. 12. The droplets can flow in channels 2270, 2271, 2272,and 2273, i.e., when valves 2293, 2294, and 2295 are in the openposition (FIG. 12A). Once the channels are filled, the flow in channels2271, 2272, and 2273 can be stopped (i.e., by closing valves 2293, 2294,and 2295) and the entrance valves to the microwells can be opened (FIG.12B). The droplets can position into the nearest microwell by surfacetension or by other forces, as discussed below. If aconcentration-dependent chemical process (e.g., crystallization) hasoccurred in a microwell, both the entrance and exit valves of thatparticular microwell can be opened while optionally keeping the othervalves closed, and a product of the concentration-dependent chemicalprocess (e.g., a crystal) can be flushed into vessel 2299, such as anx-ray capillary or a NMR tube, for further analysis.

Microwells may have any suitable size, volume, shape, and/orconfiguration, i.e., for positioning a droplet depending on theapplication. For example, microwells may have a cross-sectionaldimension of less than about 250 μm, less than about 100 μm, or lessthan about 50 μm. In some embodiments, microwells can have a volume ofless than 10 μL, less than 1 μL, less than 0.1 μL, less than 10 nL, lessthan 1 nL, less than 0.1 nL, or less than 10 pL. Microwells may have alarge volume (e.g., 0.1-10 μL) for storing large droplets, or smallvolumes (e.g., 10 pL or less) for storing small droplets.

In the embodiment illustrated in FIG. 11, microwells 2081, 2082, and2083 have the same dimensions. However, in certain other embodiments,the microwells can have different dimensions relative to one another,e.g., for holding droplets of different sizes. For instance, amicrofluidic chip can comprise both large and small microwells, wherelarge droplets may favor the large microwells and small droplets mayfavor the small microwells. By varying the size of the microwells and/orthe size of the droplets on a chip, positioning of the droplets not onlydepends on whether or not the microwell is empty, but also on whether ornot the sizes of the microwell and the droplet match. The positioning ofdifferent droplets of different sizes may be useful for varying reactionconditions within an assay.

In another embodiment, microwells 2081, 2082, and 2083 have differentshapes. For example, one microwell may be square, another may berectangular, and another may have a pyramidal shape. Different shapes ofmicrowells may allow droplets to have different surface energies whilepositioned in the microwell, and can cause a droplet to favor one shapeover another. Different shapes of microwells can also be used incombination with droplets of different size, such that droplets ofcertain sizes favor particular shapes of microwells.

Sometimes, a droplet can be released from a microwell, e.g., after areaction has occurred inside of a droplet. Different sizes, shapes,and/or configurations of microwells may influence the ability of adroplet to be released from the microwell.

In some cases, the size of the microwell is approximately the same sizeas the droplet, as shown in FIG. 11. For instance, the volume of themicrowell can be less than approximately twice the volume of thedroplet. This is particularly useful for positioning a single dropletwithin a single microwell. In other cases, however, more than onedroplet can be positioned in a microwell. Having more than one dropletin a microwell can be useful for applications that require the mergingof two droplets into one larger droplet, and for applications thatinclude allowing a component to pass (e.g., diffuse) from one droplet toanother adjacent droplet.

Although many embodiments illustrated herein show the positioning ofdroplets in microwells, in some cases, microwells are not required forpositioning droplets. For instance, in some cases, a droplet ispositioned in a region in fluid communication with the channel, theregion having a different affinity for the droplet than does anotherpart of the channel. The region may be positioned on a wall of thechannel. In one embodiment, the region can protrude from a surface(e.g., a side) of the channel. In another embodiment, the region canhave at least one dimension (e.g., a width or height) larger than adimension of the channel. A droplet that is carried in the channel maybe positioned into the region by the lowering of the surface energy ofthe droplet when positioned in the region, relative to the surfaceenergy of the droplet prior to being positioned in the region.

In another embodiment, positioning of a droplet does not require the useof differences in dimension between the region and the channel. A regionmay have a patterned surface (e.g., a hydrophobic or hydrophilic patch,a surface patterned with a specific chemical moiety, or a magneticpatch) that favors the positioning and/or containing of a droplet.Different methods of positioning, e.g., based on hydrophobic/hydrophilicinteractions, magnetic interactions, or electrical interactions such asdielectrophoresis, electrophoresis, and optical trapping, as well aschemical interactions (e.g., covalent interactions, hydrogen-bonding,van der Waals interactions, and adsorption) between the droplet and thefirst region are possible. In some cases, the region may be positionedin, or adjacent to, the channel, for example.

In some instances, a condition within a droplet can be controlled afterthe droplet has been formed. For example, FIG. 13 shows an example of amicroreactor region 2026 of device 2000 (FIG. 9). The microreactorregion can be used to control a condition in a droplet indirectly, e.g.,by changing a condition in a reservoir adjacent to a microwell ratherthan by changing a condition in the microwell directly. Region 2026includes a series of microwells used to position droplets 2201-2208, themicrowells and droplets being separated from reservoir 2140 bysemi-permeable barrier 2150. In this particular example, all dropletscontain a saline solution and are surrounded by an immiscible oil. Asshown in the figure, some droplets (droplets 2201-2204) are positionedin microwells that are farther away from the reservoir than others(droplets 2205-2208). As such, a change in a condition in reservoir 2140has a greater immediate effect on droplets 2205-2208 than on droplets2201-2204. Droplets 2201-2208 initially have the same volume inmicroreactor region 2026 (not shown).

FIGS. 13A (top view) and 13B (side view of droplets 2201, 2204, 2206,2207) show an effect that can result from circulating air in thereservoir. Air in the reservoir, in certain amounts and in connectionwith conditions that can be selected by those of ordinary skill in theart based upon this disclosure (e.g. amount, flow rate, temperature,etc. taken in conjunction with the makeup of the droplets) can causedroplets 2205-2208 to decrease in volume more than that of droplets2201-2204, since droplets 2205-2208 are positioned closer to thereservoir than droplets 2201-2204. Through the process of permeation,fluids in the droplets can move across the semi-permeable barrier,causing the volume of the droplets to decrease. As shown in FIGS. 13C(top view) and 13D (side view of droplets 2201, 2204, 2206, 2207), underappropriate conditions flowing water in the reservoir instead of airreverses this process. Small droplets 2205-2208 of FIGS. 13A and 13B canswell, as illustrated in FIGS. 13C and 13D because, for instance, thedroplets may contain a saline solution or otherwise have an appropriatedifference in osmotic potential compared to the surrounding environment.This difference in osmotic potential can cause water to diffuse from thereservoir, across the semi-permeable barrier, through the oil, and intothe droplets. Droplets farther away from the reservoir (droplets2201-2204) may initially remain small, since it takes a longer time forwater to diffuse across a longer distance (e.g., diffusion time scaleswith the square of the distance). At equilibrium, the chemicalpotentials of the fluid in the reservoir and the fluid in the dropletsgenerally will be equal.

As shown in FIG. 13, reservoir 2140 is in the form of a microfluidicchannel. In other embodiments, however, the reservoir can take ondifferent forms, shapes, and/or configurations, so long as it can beused to store a fluid. For instance, as shown in FIG. 8C, reservoir 2140is in the form of a chamber, and a series of microfluidic channels2155-1 allow fluidic access to the chamber (i.e., to introduce differentfluids into the reservoir). Sometimes, reservoirs can have componentssuch as posts 2145, which may give structured support to the reservoir.

A fluidic chip can include several reservoirs that are controlledindependently (or dependently) of each other. For instance, a device caninclude greater than 1, great than 5, greater than 10, greater than 100,greater than 1,000, or greater than 10,000 reservoirs. A large number(e.g., 100 or more) of reservoirs may be suitable for a chip in whichreservoirs and microwells are paired such that a single reservoir isused to control conditions in a single microwell. A small number (e.g.,10 or less) of reservoirs may be suitable when it is favorable for manymicrowells to experience the same changes in conditions relative to oneanother. This type of system can be used, for example, for increasingthe size of many droplets (i.e., diluting components within thedroplets) simultaneously.

Reservoir 2140 typically has at least one cross-sectional dimension inthe micron-range. For instance, the reservoir may have a length, width,or height of less than 500 μm, less than 250 μm, less than 100 μm, lessthan 50 μm, less than 10 μm, or less than 1 μm. The volume of thereservoir can also vary; for example, it may have a volume of less than50 μL, less than 10 μL, less than 1 μl, less than 100 nL, less than 10nL, less than 1 nL, less than 100 pL, or less than 10 pL. In oneparticular embodiment, a reservoir can have dimensions of 10 mm by 3 mmby 50 μm and a volume of less than 20 μL.

A large reservoir (e.g., a reservoir having a large cross-sectionaldimension and/or a large volume) may be useful when the reservoir isused to control the conditions in several (e.g., 100) microwells, and/orfor storing a large amount of fluid. A large amount of fluid in thereservoir can be useful, for example, when droplets are stored for along time (i.e., since, in some embodiments, material from the dropletmay permeate into surrounding areas or structures over time). A smallreservoir (e.g., a reservoir having a small cross-sectional dimensionand/or a small volume) may be suitable when a single reservoir is usedto control conditions in a single microwell and/or for cases where adroplet is stored for shorter periods of time.

Semi-permeable barrier 2150 is another factor that controls the rate ofequilibration or the rate of passage of a component between thereservoir and the microwells. In other words, the semi-permeable barriercontrols the degree of chemical communication between two sides of thebarrier. Examples of semi-permeable barriers include dialysis membranes,PDMS membranes, polycarbonate films, meshes, porous layers of packedparticles, and the like. Properties of the barrier that may affect therate of passage of a component across the barrier include: the materialin which the barrier is fabricated, thickness, porosity, surface area,charge, and hydrophobicity/hydrophilicity of the barrier.

The barrier may be fabricated in any suitable material and/or in anysuitable configuration in order to permit one set of components andinhibit another set of components from crossing the barrier. In oneembodiment, the semi-permeable barrier comprises the material from whichthe reservoir is formed, i.e., as part of layer 2149 as shown in FIG.8C, and can be formed in the same process in which the reservoir isformed (i.e., the reservoir and the barrier can be formed in a singleprocess in which a precursor fluid is spin-coated or solution-cast ontoa mold and subsequently hardened to form both the barrier and reservoirin a single step, or, alternatively, another process in which thebarrier and reservoir are formed from the same material, optionallysimultaneously). In another embodiment, the semi-permeable barriercomprises the same material as the structure of the device, i.e., aspart of structure 2135 as shown in FIG. 8D, and can be formed inconjunction with the structure 2135 as described above in connectionwith the semi-permeable barrier and reservoir, optionally. For instance,all, or a portion of, the barrier can be formed in the same material asthe structure layer and/or reservoir layer. In some cases, the barriercan be fabricated in a mixture of materials, one of the materials beingthe same material as the structure layer and/or reservoir layer.Fabricating the barrier in the same material as the structure layerand/or reservoir layer offers certain advantages such as easyintegration of the barrier into the device. In other embodiments, thesemi-permeable barrier is fabricated as a layer independent of thestructure layer and reservoir layer. The semi-permeable barrier can bemade in the same or a different material than the other layers of thedevice.

In some cases, the barrier is fabricated in a polymer (e.g., a siloxane,polycarbonate, cellulose, etc.) that allows passage of a first set oflow molecular weight components, but inhibits the passage of a secondset of large molecular weight components across the barrier. Forinstance, a first set of low molecular weight components may includewater, gases (e.g., air, oxygen, and nitrogen), water vapor (e.g.,saturated or unsaturated), and low molecular weight organic solvents(e.g., hexadecane), and the second set of large molecular weightcomponents may include proteins, polymers, amphiphiles, and/or othersspecies. Those of ordinary skill in the art can readily select asuitable material for the barrier based upon e.g., its porosity, itsrigidity, its inertness to (i.e., freedom from degradation by) a fluidto be passed through it, and/or its robustness at a temperature at whicha particular device is to be used.

The semi-permeable barrier may have any suitable thickness for allowingone set of components to pass across the barrier while inhibitinganother set of components. For example, a semi-permeable barrier mayhave a thickness of less than 10 mm, less than 1 mm, less than 500 μm,less than 100 μm, less than 50 μm, or less than 20 μm, or less than 1μm. A thick barrier (e.g., 10 mm) may be useful for allowing slowpassage of a component between the reservoir and the microwell. A thinbarrier (e.g., less than 20 μm thick) can be used when it is desirablefor a component to be passed quickly across the barrier.

For size exclusive semi-permeable barriers (i.e., including dialysismembranes), the pores of the barriers can have different shapes and/orsizes. In one embodiment, the sizes of the pores of the barrier arebased on the inherent properties of the barrier, such as the degree ofcross-linking of the material in which the barrier is fabricated. Inanother embodiment, the pores of the barrier are machine-fabricated in afilm of a material. Semi-permeable barriers may have pores sizes of lessthan 100 μm, less than 10 μm, less than 1 μm, less than 100 nm, lessthan 10 nm, or less than 1 nm, and may be chosen depending on thecomponent to be excluded from crossing the barrier.

A semi-permeable barrier may exclude one or more components from passingacross it by methods other than size-exclusion, for example, by methodsbased on charge, van der Waals interactions, hydrophilic or hydrophobicinteractions, magnetic interactions, and the like. For instance, thebarrier may inhibit magnetic particles but allow non-magnetic particlesto pass across it (or vice versa).

Different methods of passing a component across the semi-permeablebarrier can be used. For instance, in one embodiment, the component maydiffuse across the barrier if there is a difference in concentration ofthe component between the micro well and the reservoir. In anotherembodiment, if the component is water, water can pass across the barrierby osmosis. In yet another embodiment, the component can evaporateacross the barrier; for instance, a fluid in the microwell can evaporateacross the barrier if a gas is positioned in the reservoir. In somecases, the component can cross the barrier by bulk or mass flow inresponse to a pressure gradient in the microwell or the reservoir. Inother cases, the component can cross the barrier by methods such asfacilitated diffusion or by active transport. A combination of modes oftransport can also be applied. Typically, however, the barrier is notconstructed and arranged to be operatively opened and closed to permitand inhibit fluid flow in the reservoir, microwell, or microchannel. Forinstance, in one embodiment, the barrier does not act as a valve thatcan operatively open and close to allow and block, respectively, fluidicaccess to the reservoir, microwell, or microchannel.

In some cases, the barrier is positioned in a device such that fluid canflow adjacent to a first side of the barrier without the need for thefluid to flow through the barrier. For instance, in one embodiment, abarrier is positioned between a reservoir and a microwell; the reservoirhas an inlet and an outlet that allow fluidic access to it, and themicrowell is fluidically connected to a microchannel having an inlet andan outlet, which allow fluidic access to the microwell. Fluid can flowin the reservoir without necessarily passing across the barrier (i.e.,into the microchannel and/or microwell), and the same or a differentfluid can flow in the microchannel and/or microwell without necessarilypassing across the barrier (i.e., into the reservoir).

FIG. 14 shows that device 2010 can be used to grow, and control thegrowth of, a precipitate such as crystal inside a microwell of thedevice. In this particular embodiment, droplet 2079 is aqueous andcontains a mixture of components, e.g., a protein, a salt, and a buffersolution, for generating a crystal. The components are introduced intothe device via inlets 2050, 2055, and/or 2060. An immiscible oilintroduced into inlets 2045 and 2065 serves as carrier fluid 2048. Asshown schematically in FIG. 14B, droplet 2079 is surrounded by carrierfluid 2048 in microwell 2130. Semi-permeable barrier 2150 separates themicrowell from reservoir 2140, which can contain posts 2145.

Protein in droplet 2079 can be nucleated to form crystal 2300 byconcentrating the protein solution within the droplet (FIG. 14C). If theprotein solution is concentrated to a certain degree, the solutionbecomes supersaturated and suitable for crystal growth. In oneembodiment, the protein solution is concentrated by flowing air inreservoir 2150, which causes water in the droplet to evaporate acrossthe semi-permeable barrier while the protein remains in the droplet. Inanother embodiment, a high ionic strength buffer (i.e., a buffer havinghigher ionic strength than the ionic strength of the fluid defining thedroplet) is flowed in the reservoir. The imbalance of chemical potentialbetween the two solutions causes water to diffuse from the droplet toreservoir. Other methods for concentrating the solution within thedroplet can also be used. Other methods for nucleating a crystal canalso be applied. For instance, two droplets, each of which contain acomponent necessary for protein crystallization, can be positioned in asingle microwell. The two droplets can be fused together into a singledroplet, i.e., by changing the concentration of surfactant in thedroplets, thereby causing the components of the two droplets to mix. Insome cases, these conditions may be suffice to cause nucleation.

As shown in FIGS. 14C and 14D, once crystal 2300 is nucleated in adroplet, the crystal grows spontaneously within a short period of time(e.g., 10 seconds) since the crystal is surrounded by a supersaturatedsolution (as discussed in more detail below). In some cases, this rapidgrowth of the crystal leads to poor-quality crystals, since defects donot have time to anneal out of the crystal. One solution to this problemis to change the conditions of the sample during the crystallizationprocess. Ideal crystal growing conditions occur when the sample istemporarily brought into deep supersaturation where the nucleation rateis high enough to be tolerable. In the ideal scenario, after a crystalhas nucleated, the supersaturation of the solution would be decreased,e.g., by lowering the protein or salt concentrations or by raisingtemperature, in order to suppress further crystal nucleation and toestablish conditions where slow, defect free crystal growth occurs.Device 2010 can allow this process to occur by decreasing the size of acrystal after it has nucleated and grown, and then re-growing thecrystal slowly under moderately supersaturated conditions. Thus, theprocesses of nucleation and growth can be performed reversibly, and canoccur independently of each other, in embodiments such as device 2010.

To decrease the size of the crystal (i.e., so that the crystal can bere-grown to become defect-free), reservoir 2140 can be filled with abuffer of lower salt concentration than that of the protein solution inthe droplet. This causes water to flow in the opposite direction, i.e.,from the reservoir to the protein solution, which dilutes the proteinand the precipitant (e.g., by increasing the volume of the droplet),suppresses further nucleation, and slows down growth (FIG. 14E). Tore-grow the crystal under slower and more moderately supersaturatedconditions, the fluid in the reservoir can be replaced by a solutionhaving a higher salt concentration such that fluid diffuses slowly outof the droplet, thereby causing the protein in the droplet toconcentrate.

If the dialysis step of decreasing the size of the crystal proceeds longenough that the crystal dissolves completely, this system (e.g., device2010) can advantageously allow the processes of nucleation and growth tobe reversed, i.e., by changing the fluids in the reservoir. In addition,if small volumes of the droplets (e.g., ˜nL) are used in this system,the device allows faster equilibration times between the droplet and thereservoir than for microliter-sized droplets, which are used inconventional vapor diffusion-based crystallization techniques (e.g.,hanging or sitting drop techniques).

In some cases, concentrating the protein solution within the dropletcauses the nucleation of precipitate (FIG. 14F). The precipitate maycomprise largely noncrystalline material, largely crystalline material,or a combination of both non-crystalline and crystalline material,depending on the growth conditions applied. Device 2010 can be used todilute the protein solution in the droplet, which can cause some, orall, of the precipitate to dissolve. Sometimes, the precipitate isdissolved until a small portion of the precipitate remains. Forinstance, dissolving may cause the smaller portions of the precipitateto dissolve, allowing one or a few of the largest portions to remain;these remaining portions can be used as seeds for growing crystals.After a seed has been formed, the concentration of protein in thedroplet can be increased slowly (e.g., by allowing water to diffuseslowly out of the droplet). This process can allow the formation oflarge crystals within the droplet (FIG. 14G).

As shown in FIGS. 14A-14G, processes such as nucleation, growth, anddissolution of a crystal can all occur within a droplet while thedroplet is positioned in the same microwell. In other embodiments,however, different processes can occur in different parts or regions ofthe fluidic network. For instance, nucleation and dissolution of acrystal can take place in a small (e.g., 10 pL) droplet in a smallmicrowell, and then the droplet containing the crystal can betransported to a larger microwell for re-growth of the crystal in alarger (e.g., 1 nL) droplet. This process may allow small amounts ofreagent to be consumed for the testing of reaction conditions and largeramounts of reagent to be used when reaction conditions are known. Insome cases, this process decreases the overall amount of reagentconsumed, as discussed in more detail below.

Device 2010 of FIG. 15 can be used to form many droplets of differentcomposition, and to precisely control the rate and duration ofsupersaturation of the protein solution within each droplet. The rate ofintroduction of protein, salt, and buffer solutions into inlets 2050,2055, and 2060 can be varied so that the solutions can becombinatorially mixed with each other to produce several (e.g., 1000)droplets having different chemical compositions. In one embodiment, eachdroplet has the same volume (e.g., 2 nL), and each droplet can contain,for instance, 1 nL of protein solution and 1 nL of the other solutes.The rate of introducing the protein solution can be held constant, whilethe rates of introducing the salt and buffer solutions can vary. Forexample, injection of the salt solution can ramp up linearly in time(e.g., from 0 to 10 nL/s), while injection of the buffer solution rampsdown linearly in time (e.g., from 10 to 0 nL/s). In another embodiment,the rate of introducing a protein can vary while one of the othersolutes is held constant. In yet another embodiment, all solutionsintroduced into the device can be varied, i.e., in order to makedroplets of varying sizes. Advantageously, this setup can allow manydifferent conditions for protein crystallization to be testedsimultaneously.

In addition to varying the concentration of solutes within each droplet,the environmental factors influencing crystallization can be changed.For instance, device 2010 includes five independent reservoirs 2140-1,2140-2, 2140-3, 2140-4, and 2140-5 that can contain solutions ofdifferent chemical potential. These reservoirs can be used to vary thedegree of supersaturation of the protein solution within the droplets.Thus, the nucleation rate of the first crystal produced and the growthrate of the crystal can be controlled precisely within each droplet.Examples of controlling the sizes of crystals are shown in FIGS. 15B and15 C, and in Example 3.

FIG. 16B is a phase diagram illustrating the use of a reservoir tochange a condition in a droplet (i.e., by reversible dialysis). At lowprotein concentrations, a protein solution is thermodynamically stable(i.e., in the stable solution phase). An increase in concentration of aprecipitant, such as salt or poly(ethylene)glycol (PEG), drives theprotein into a region of the phase diagram where the solution ismetastable and protein crystals are stable (i.e., in the co-existencephase). In this region, there is a free energy barrier to nucleatingprotein crystals and the nucleation rate can be extremely slow (FIG.16A). At higher concentrations, the nucleation barrier is suppressed andhomogeneous nucleation occurs rapidly (i.e., in the crystal phase). Asmentioned above, at high supersaturation, crystal growth is rapid anddefects may not have time to anneal out of the crystal, leading to poorquality crystals. Thus, production of protein crystals requires twoconditions that work against each other. On one hand, highsupersaturation is needed for nucleating crystals, but on the otherhand, low supersaturation is necessary for crystal growth to proceedslowly enough for defects to anneal away. Changing sample conditionsduring the crystallization process is one method for solving thisproblem. Ideal crystal growing conditions occur when the sample istemporarily brought into deep supersaturation where the nucleation rateis high enough to be tolerable. In the ideal scenario, after a fewcrystals have nucleated, the supersaturation of the solution would bedecreased by either lowering the protein or salt concentrations, or byraising temperature in order to suppress further crystal nucleation andto establish conditions where slow, defect free crystal growth occurs.In other words, independent control of nucleation and growth is desired.

As shown in FIG. 16B, a microfluidic device (e.g., device 2010) of thepresent invention can be used to independently control nucleation andgrowth of a crystal. In FIG. 16B, lines 2400 and 2401 separate theliquid-crystal phase boundary. Dashed tie-lines connect co-existingconcentrations, with crystals high in protein and low in precipitant(e.g., polyethylene glycol (PEG)). For clarity, the compositiontrajectory for one initial condition is shown here, while FIG. 17 showstrajectories for multiple initial and final conditions. Reversiblemicrodialysis can be shown in three steps. Step 1: Initialconcentrations of solutions in the droplets are stable solutions(circles 2405—points a). Step 2: Dialysis against high salt or air(e.g., in the reservoir) removes water from the droplet, concentratingthe protein and precipitant within the droplet (path a→b). At point b,the solution is metastable and if crystals nucleate, then phaseseparation occurs along tie-lines (b→b′), producing small crystals thatgrow rapidly. Step 3: Dialysis against low salt water dilutes theprotein and precipitant within the droplets, which lowers Δμ andincreases ΔG* and r*. This suppresses further nucleation, causes thesmall crystals to dissolve adiabatically along the equilibrium phaseboundary (b′→c′), and slows the growth of the remaining large crystals.If there was no nucleation at point b, then the metastable solutionwould evolve from b→c. Step 4: If necessary, crystalline defects can beannealed away by alternately growing and shrinking individual crystalsb′

c′ which is accomplished by appropriately varying the reservoirconditions.

The size of a crystal that has been formed in a droplet can vary (i.e.,using device 2010 of FIG. 15). For example, a crystal may have a lineardimension of less than 500 μm, less than 250 μm, less than 100 μm, lessthan 50 μm, less than 25 μm, less than 10 μm, or less than 1 μm. Some ofthese crystals can be used for X-ray diffraction and for structuredetermination. For instance, consider the crystals formed in 1 nLdroplets. If the concentration of the protein solution introduced intothe device is 10 mg/mL=10 μg/μL, then 1 μL of protein solution onlycontains 10 μg of protein. In the device, 1 μL of protein solution canproduce 1,000 droplets of different composition, for example, eachdroplet containing 1 nL of protein solution and 1 nL of other solutes,as described above. The linear dimension of a 1 nL drop is 100 μm and ifthe crystal is 50% protein, then the crystal will have a volume 50 timessmaller than the protein solution, or 20 pL. The linear dimension of acubic crystal of 20 pL volume is roughly 25 μm, and X-ray diffractionand structure determination from such small crystals is possible.

In another embodiment, a device having two sections can be used to formcrystals. The first section can be used to screen for crystallizationconditions, for instance, using very small droplet volumes (e.g., 50pL), which may be too small for producing protein crystals for X-raydiffraction and for structure determination. Once favorable conditionshave been screened and identified, the protein stock solution can bediverted to a second section designed to make droplets of larger size(e.g., 1 nL) for producing crystals suitable for diffraction. Using sucha device, screening, e.g., 1000 conditions at 50 pL per screen, consumesonly 0.5 μg of protein. Scaling up a subset of 50 conditions to 1 nL(e.g., the most favorable conditions for crystallization) consumesanother 0.5 μg of protein. Thus, it can be possible to screen 1000conditions for protein crystallization using a total of 1 μg of protein.

In some cases, it is desirable to remove the proteins formed within themicrowells of the device, for instance, to load them into vessels suchas x-ray capillaries for performing x-ray diffraction, as shown in FIG.12. In one embodiment, a microfluidic device comprises microwells thatare connected to an exhaust channel and a valve that controls thepassage of components from the microwell to the exhaust channel. Usingthe multiplexed valves, it is possible to control n valves with 2 log₂ npressure lines used to operate the valves. Droplets can first be loadedinto individual microwells using surface tension forces as describeabove. Then, individual microwells can be addressed in arbitrary order(e.g., as in a random access memory (RAM) device) and crystals can bedelivered into x-ray capillaries. Many (e.g., 100) crystals, eachisolated from the next by a plug of immiscible fluid (e.g.,water-insoluble oil), can be loaded into a single capillary fordiffraction analysis.

As the number of crystallization trials grows, it may be advantageous toautomate the detection of crystals. In one embodiment, commercial imageprocessing programs that are interfaced to optical microscopes equippedwith stepping motor stages are employed. This software can identify andscore “hits” (e.g., droplets and conditions favorable for proteincrystallization). This subset of all the crystallization trials can bescanned and select crystals can be transferred to the x-ray capillary.

In another embodiment, a microfluidic device has a temperature controlunit. Such a device may be fabricated in PDMS bonded to glass, or toindium tin oxide (ITO) coated glass, i.e., to improve thermalconductivity. Two thermoelectric devices can be mounted on oppositesides of the glass to create a temperature gradient. Thermoelectricdevices can supply enough heat to warm or cool a microfluidic device atrates of several degrees per minute over a large temperature range.Alternatively, thermoelectric devices can maintain a stable gradientacross the device. For example, device 2010 shown in FIG. 15A can have athermoelectric device set at 40° C. on the left end (i.e., nearreservoir 2140-1) and at 4° C. on the right end (i.e., near reservoir2140-5). This arrangement can enable each of the reservoirs in betweenthe left and right ends to reside at different temperatures. Temperaturecan be used as a thermodynamic variable, in analogy to concentration inFIG. 16B, to help decouple nucleation and growth.

In some cases, surfactants are required to prevent coalescence ofdroplets. For instance, in one embodiment, several droplets can bepositioned adjacent to each other in a channel without the use ofmicrowells, i.e., the droplets can line themselves in differentarrangements along the length of the channel. In this embodiment, aswell as embodiments that involve the passing of droplets beside otherdroplets (FIG. 11), a surfactant is required to stabilize the droplets.For each type of oil (i.e., used as a carrier fluid), there exists anoptimal surfactant (i.e., an optimum oil/surfactant pair). For example,for a device that is fabricated in PDMS, the ideal pair includes asurfactant that stabilizes an aqueous droplet and does not denature theprotein, and an oil that is both insoluble in PDMS, and has a watersolubility similar to PDMS. Hydrocarbon-based oils such as hexadecaneand dichloromethane can be poor choices, since these solvents swell anddistort the PDMS device after several hours. The best candidates may befluorocarbons and fluorosurfactants to stabilize the aqueous solutionbecause of the low solubility of both PDMS and proteins in fluorinatedcompounds. The use of a hydrocarbon surfactant to stabilize proteindroplets could interfere with membrane protein crystallization ofprotein-detergent complexes, although it is also possible thatsurfactants used in the protein-detergent complex also stabilizes theoil/water droplets. In one embodiment, hexadecane is used to createaqueous droplets with a gentle non-ionic detergent (e.g., Span-80) tostabilize the droplets. After the droplets are stored in the microwells,the hexadecane and Span-80 can be flushed out and replaced withfluorocarbon or paraffin oil. This process can allow the hexadecane toreside in the PDMS for a few minutes, which is too short of a time todamage the PDMS device.

In another embodiment, the droplet-stabilizing surfactant can beeliminated by having a device in which there are no microwells, andwhere the protein droplets are separated in a microchannel by plugs ofan oil. For a device that is fabricated in a polymer such as PDMS, anoil separating the protein droplets may dissolve into the bulk of thepolymer device over time. This can cause the droplets to coalescebecause the droplets are not stabilized by a surfactant. In some cases(e.g., if an oil that is insoluble in the polymer cannot be found and/orif coalescence of droplets is not desired), the microfluidic structurecontaining the protein channels can be made from glass, and the barriersand valves can be made in a polymer (e.g., PDMS). Because the volume ofthe barrier is less than the volume of oil, only a small fraction of theoil can dissolve into the barrier, causing the aqueous droplets toremain isolated.

The device described above (i.e., without microwells, and where theprotein droplets are separated in a microchannel by plugs of oil) may beused to control the nucleation and growth of crystals similar to that ofdevice 2010. For instance, a semi-permeable barrier can separate themicrochannel from a reservoir, and fluids such as air, vapor, water, andsaline can be flowed in the reservoir to induce diffusion of wateracross the barrier. Therefore, swelling and shrinking of the droplet,and the formation and growth of crystals within the droplet, can becontrolled.

FIG. 18 shows another example of a device that can be used to enable aconcentration-dependent chemical process (e.g., crystallization) tooccur. Device 2500 includes a microwell 2130 fluidically connected tomicrochannel 2125. Beneath the microwell are reservoirs 2140 and 2141(e.g., in the form of microchannels, which may be connected orindependent), separated by semi-permeable barrier 2150. Droplet 2079(e.g., an aqueous droplet) may be positioned in the microwell,surrounded by an immiscible fluid (e.g., an oil), as shown in FIG. 18C.In some cases, dialysis processes similar to ones described above can beimplemented. For example, fluids can be transported across thesemi-permeable barrier by various methods (e.g., diffusion orevaporation) to change the concentration and/or volume of the fluid inthe droplet.

In other cases, a vapor diffusion process can occur in device 2500. Forinstance, a portion of the oil that is used as a carrier fluid inmicrochannel 2125 can be blown out of the channel with a fluid such as agas (e.g., dry air or water saturated air) by flowing the gas into aninlet of the channel. This process can be performed while the dropletremains in the microwell (FIG. 18D). Depending on the chemical potentialof the gas in the channel, the droplets containing protein canconcentrate or dilute. For example, if air is flowed into microchannel2125, water from the droplet can exchange (e.g., by evaporation) out ofthe droplet and into the air stream. This causes the droplet to shrinkin volume (FIG. 18E). To dilute the protein in the droplet and/or toincrease the volume of the droplet, a stream of saturated water vaporcan be flowed into microchannel 2125 (FIG. 18F).

In another embodiment, concentration-dependent chemical processes canoccur in a device without the use of droplets. For instance, a firstfluid can be positioned in a region of the fluidic network (e.g., in amicrowell) and a second fluid can be positioned in a reservoir, theregion and the reservoir separated by a semi-permeable barrier. Theintroduction of different fluids into the reservoir can cause a changein the concentration of components within the first region, i.e., bydiffusion of certain components across the semi-permeable barrier.

To overcome the “‘world to chip’ interface problem” of introducing aprotein solution into a microfluidic device without wasting portions ofthe protein solution, e.g., in connections or during the initial purgingof air from the microfluidic device, devices of the present inventioncan be fabricated with an on-chip injection-loop system. For example,buffer region 2022 of FIG. 9 with its neighboring valves (e.g., valves2093 and 2100) can function as an injection-loop if it is locatedupstream from the nozzle (i.e., upstream of intersection 2075). A volume(e.g., of protein solution can first be dead-end loaded into a longchannel (e.g., having dimensions 100 mm×0.1 mm×0.1 mm) and then isolatedwith valves. Next, the device can be primed and purged of air. Oncedroplets are being produced steadily, the injection-loop can beconnected fluidically to the flow upstream from the nozzle by theactuation of valves.

In some embodiments, regions of a fluidic network such as microchannelsand micro wells are defined by voids in the structure. A structure canbe fabricated of any material suitable for forming a fluidic network.Non-limiting examples of materials include polymers (e.g., polystyrene,polycarbonate, PDMS), glass, and silicon. Those of ordinary skill in theart can readily select a suitable material based upon e.g., itsrigidity, its inertness to (i.e., freedom from degradation by) a fluidto be passed through it, its robustness at a temperature at which aparticular device is to be used, its hydrophobicity/hydrophilicity,and/or its transparency/opacity to light (i.e., in the ultraviolet andvisible regions).

In some instances, a device is comprised of a combination of two or morematerials, such as the ones listed above. For instance, the channels ofthe device may be formed in a first material (e.g., PDMS), and asubstrate can be formed in a second material (e.g., glass). In oneparticular example as shown in FIG. 8, structure 2135, which containsvoids in the form of channels and microwells, can be made in PDMS,support layer 2136 can be made in PDMS, and support layer 2137 may beformed in glass.

Most fluid channels in components of the invention have maximumcross-sectional dimensions less than 2 mm, and in some cases, less than1 mm. In one set of embodiments, all fluid channels containingembodiments of the invention are microfluidic or have a largest crosssectional dimension of no more than 2 mm or 1 mm. In another embodiment,the fluid channels may be formed in part by a single component (e.g., anetched substrate or molded unit). Of course, larger channels, tubes,chambers, reservoirs, etc. can be used to store fluids in bulk and todeliver fluids to components of the invention. In one set ofembodiments, the maximum cross-sectional dimension of the channel(s)containing embodiments of the invention are less than 500 microns, lessthan 200 microns, less than 100 microns, less than 50 microns, or lessthan 25 microns. In some cases the dimensions of the channel may bechosen such that fluid is able to freely flow through the article orsubstrate. The dimensions of the channel may also be chosen, forexample, to allow a certain volumetric or linear flowrate of fluid inthe channel. Of course, the number of channels and the shape of thechannels can be varied by any method known to those of ordinary skill inthe art. In some cases, more than one channel or capillary may be used.For example, two or more channels may be used, where they are positionedinside each other, positioned adjacent to each other, positioned tointersect with each other, etc.

A “channel,” as used herein, means a feature on or in an article(substrate) that at least partially directs the flow of a fluid. Thechannel can have any cross-sectional shape (circular, oval, triangular,irregular, square or rectangular, or the like) and can be covered oruncovered. In embodiments where it is completely covered, at least oneportion of the channel can have a cross-section that is completelyenclosed, or the entire channel may be completely enclosed along itsentire length with the exception of its inlet(s) and outlet(s). Achannel may also have an aspect ratio (length to average cross sectionaldimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 ormore. An open channel generally will include characteristics thatfacilitate control over fluid transport, e.g., structuralcharacteristics (an elongated indentation) and/or physical or chemicalcharacteristics (hydrophobicity vs. hydrophilicity) or othercharacteristics that can exert a force (e.g., a containing force) on afluid. The fluid within the channel may partially or completely fill thechannel. In some cases where an open channel is used, the fluid may beheld within the channel, for example, using surface tension (i.e., aconcave or convex meniscus).

The channels of the device may be hydrophilic or hydrophobic in order tominimize the surface free energy at the interface between a materialthat flows within the channel and the walls of the channel. Forinstance, if the formation of aqueous droplets in an oil is desired, thewalls of the channel can be made hydrophobic. If the formation of oildroplets in an aqueous fluid is desired, the walls of the channels canbe made hydrophilic.

In some cases, the device is fabricated using rapid prototyping and softlithography. For example, a high resolution laser printer may be used togenerate a mask from a CAD file that represents the channels that makeup the fluidic network. The mask may be a transparency that may becontacted with a photoresist, for example, SU-8 photoresist (MicroChem),to produce a negative master of the photoresist on a silicon wafer. Apositive replica of PDMS may be made by molding the PDMS against themaster, a technique known to those skilled in the art. To complete thefluidic network, a flat substrate, e.g., a glass slide, silicon wafer,or a polystyrene surface, may be placed against the PDMS surface andplasma bonded together, or may be fixed to the PDMS using an adhesive.To allow for the introduction and receiving of fluids to and from thenetwork, holes (for example 1 millimeter in diameter) may be formed inthe PDMS by using an appropriately sized needle. To allow the fluidicnetwork to communicate with a fluid source, tubing, for example ofpolyethylene, may be sealed in communication with the holes to form afluidic connection. To prevent leakage, the connection may be sealedwith a sealant or adhesive such as epoxy glue.

In order to optimize a device of the present invention, it may behelpful to quantify the diffusion constant and solubility of certainfluids through the semipermeable barrier, if these quantities are notalready known. For instance, if the barrier is fabricated in PDMS, theflux of water through the barrier can be quantified by measuringtransport rates of water as a function of barrier thickness. Microfluidic devices can be built to have a well-defined planar geometriesfor which analytical solutions to the diffusion equation are easilycalculated. For example, a microfluidic device can be fabricated havinga 2 mm by 2 mm square barrier separating a water-filled chamber from achamber through which dry air flows. The flux can be measured by placingcolloids in the water and measuring the velocity of the colloids as afunction of time. Analysis of the transient and steady-state flux allowsdetermination of the diffusion constant and solubility of water in PDMS.Similar devices can be used to measure the solubility of oil in PDMS. Inorder to optimize the reversible dialysis process, the flux of waterinto and out of the protein solutions in the droplets can be determined(e.g., as a function of droplet volume, ionic strength of the fluids inthe reservoir and/or droplet, type of carrier oil, and/or thickness ofthe barrier) using video optical microscopy by measuring the volume ofthe droplets as a function of time after changing the solution in thereservoir.

The present invention is not limited by the types of proteins that canbe crystallized. Examples of types of proteins includebacterially-expressed recombinant membrane channel proteins, Gprotein-coupled receptors heterologously expressed in a mammalian cellculture systems, membrane-bound ATPase, and membrane proteins.

Microfluidic methods have been used to screen conditions for proteincrystallization, but until now this method has been applied mainly toeasily handled water-soluble proteins. A current challenge in structuralbiology is the crystallization and structure determination of integralmembrane proteins. These are water-insoluble proteins that reside in thecell membrane and control the flows of molecules into and out of thecell. They are primary molecular players in such central biologicalphenomena as the generation of electrical impulses in the nervoussystem, “cell signaling,” i.e., the ability of cells to sense andrespond to changes in environment, and the maintenance of organismalhomeostasis parameters such as water and electrolyte balance, bloodpressure, and cytoplasmic ATP levels. Despite their vast importance inmaintaining cell function and viability, membrane proteins (which makeup roughly 30% of proteins coded in the human genome) areunder-represented in the structural database (which contains >10⁴water-soluble proteins and <10² membrane proteins). The reason for thisscarcity is because it has been difficult to express membrane proteinsin quantities large enough to permit crystallization trials, and evenwhen such quantities are available, crystallization itself is notstraight-forward.

Devices of the present invention may be used to exploit recent advancesin membrane protein expression and crystallization strategies. Forinstance, some expression systems for prokaryotic homologues ofneurobiologically important eukaryotic membrane proteins have beendeveloped, and in a few cases these have been crystallized andstructures determined by x-ray crystallography. In these cases, however,the rate-limiting step, is not the production of milligram-quantities ofprotein, but the screening of crystallization conditions. Membraneproteins must be crystallized from detergent solutions, and the choiceand concentration of detergent have been found to be crucial additionalparameters in finding conditions to form well-diffracting crystals. Forthis reason, a typical initial screen for a membrane protein requiressystematic variation of 100-200 conditions. Sparse-matrix screens simplydon't work because they are too sparse. Moreover, two additionalconstraints make the crystallization of membrane proteins more demandingthan that of water-soluble proteins. First, the amounts of proteinobtained in a typical membrane protein preparation, even in the best ofcases, are much smaller than what is typically encountered inconventional water-soluble proteins (i.e., 1-10 mg rather than 50-500mg). Second, membrane proteins are usually unstable in detergent andmust be used in crystallization trials within hours of purification;they cannot be accumulated and stored. These constraints run directlyagainst the requirement for large, systematic crystal screens.

Devices of the present invention may be used to overcome the constraintsmentioned above for crystallizing membrane proteins. For example, device2010, which can be used to perform reversible dialysis, may overcome thethree limitations of membrane protein crystallization: the small amountof protein available, the short time available to handle the pureprotein, and the very large number of conditions that must be tested tofind suitable initial conditions for crystallization.

One of the challenges of crystallography is for the growth of extremelyordered and in some cases, large, crystals. Ordered and large crystalsare suitable for ultra-high resolution data and for neutron diffractiondata, respectively. These two methods are expected to provide thelocations of protons, arguably the most important ions in enzymology,which are not accessible by conventional crystallography. So far, theseapplications have relied on serendipitous crystal formation rather thanon controlled formation of crystals. Routine access of such orderedand/or large would make structural enzymology and its applications,e.g., drug design, more powerful than it is today. Certain embodimentsof the current invention, with their ability to reversibly varysupersaturation, can be used to grow single crystals to large sizes, andthe diffraction quality of these crystals can be characterized.

Although devices and methods of the present invention have been mainlydescribed for crystallization, devices and methods of the invention mayalso be used for other types of concentration-dependent chemicalprocesses. Non-limiting examples of such processes include chemicalreactions, enzymatic reactions, immuno-based reactions (e.g.,antigen-antibody), and cell-based reactions.

The following examples are intended to illustrate certain embodiments ofthe present invention, but are not to be construed as limiting and donot exemplify the full scope of the invention.

Example 1

This example illustrates a procedure for fabricating a microfluidicstructure used in certain embodiments of the invention. In oneembodiment, a microfluidic structure comprising a series of microfluidicchannels and microwells was made by applying a standard molding articleagainst an appropriate master. For example, microchannels were made inPDMS by casting PDMS prepolymer (Sylgard 184, Dow Corning) onto apatterned photoresist surface relief (a master) generated byphotolithography. The pattern of photoresist comprised the channels andmicrowells having the desired dimensions. After curing for 2 h at 65° C.in an oven, the polymer was removed from the master to give afree-standing PDMS mold with microchannels and microwells embossed onits surface. Inlets and/or outlets were cut out through the thickness ofthe PDMS slab using a modified borer.

A semi-permeable membrane (15 microns thick) formed in PDMS andcomprising a reservoir and valve, as illustrated in FIG. 8C, wasfabricated via spin coating PDMS prepolymer onto a master generated byphotolithography. The master comprised a pattern of photoresistcomprising the reservoir and valve having the desired dimensions. Themembrane layer was cured for 1 h at 65° C. in an oven.

Next, the PDMS mold and PDMS membrane layer were sealed together byplacing both pieces in a plasma oxidation chamber and oxidizing them for1 minute. The PDMS mold was then placed onto the membrane layer with thesurface relief in contact with the membrane layer. A irreversible sealformed as a result of the formation of bridging siloxane bonds (Si—O—Si)between the two substrates, caused by a condensation reaction betweensilanol (SiOH) groups that are present at both surfaces after plasmaoxidation. After sealing, the membrane layer (with the attached PDMSmold) was removed from the master. The resulting structure was thenplaced against a support layer of PDMS. This example illustrates that amicrofluidic structure comprising microchannels, microwells, reservoirs,and valves can be fabricated using simple lithographic proceduresaccording to one embodiment of the invention.

Example 2

FIG. 10B shows the use of colloids to test combinatorial mixing ofsolutes and to visualize fluid flow using a microfluidic structure asgenerally illustrated in FIG. 9, which was made by the proceduresgenerally described in Example 1. The colloid particles, 1 μm in sizewith a size variation of 2.3%, were made by Interfacial DynamicCorporation. The concentration of the colloids was about 1%. The colloidsuspension and buffer solution were flowed into inlets 2050 and 2060,respectively, using syringes connected to a syringe pump made by HarvardApparatus, PHD2000 Programmable. The colloids were mixed with buffer bylinearly varying the flow rates of the colloid suspension and buffersolution; for instance, the flow rate of the colloidal suspension waslinearly and repeatedly varied from 80 μl/hr to 20 μl/hr while the flowrate of the buffer solution was linearly and repeatedly varied from 20μl/hr to 80 μl/hr. This was performed so that the total flow rate of theaqueous suspension was kept constant at lOO μl/hr, and so that the dropsize remained constant. The transmitted light intensity through thedroplets was proportional to the colloid concentration. The transmittedlight intensity was measured by estimating the gray scale of dropletsshown in pictures taken by a high speed camera, Phantom V5. The pictureswere taken at a rate of 10,000 frames per second. The gray scaleestimation was performed using Image-J software. This experiment showsthat combinatorial mixing of solutes can be used to generate many (e.g.,1000) different reaction conditions, each droplet being unique to aparticular condition.

Example 3

This example shows the control of droplet size within microwells of adevice. Experiments were performed using a microfluidic structure asgenerally illustrated in FIG. 13, which was made according to theprocedures generally described in Example 1. AU microwells were 200 μmwide and 30 μm in height, and the initial diameter of the droplets whilethe droplets were stored in the microwells was about 200 μm. Aqueousdroplets comprised a IM, NaCl solution. The droplets flowed in a movingcarrier phase of PFD (perfluorodecalin, 97%, Sigma-Aldrich). All fluidswere injected into device 2026 using syringe pumps (Harvard Apparatus,PHD2000 Programmable).

Device 2026 of FIG. 13 contained two sets of microwells for holdingaqueous droplets. One set of microwells contained droplets of proteinsolution (droplets 2205-2208) that were separated from the reservoir bya 15 μm thick PDMS membrane that was permeable to water, but not tosalt, PEG, or protein. Droplets in these microwells changed theirvolumes rapidly in contrast to droplets in microwells that were located100 μm away from the reservoir (e.g., droplets 2201-2204). In FIG. 13,the process of fluid exchange between the reservoir and the microwellswas diffusive, and diffusion time scales with the square of thedistance. Thus, the time to diffuse 100 μm was 44 times longer than thetime to diffuse 15 μm.

Initially, all the droplets in FIG. 13 A were of the same size andvolume. Dry air was circulated in the reservoir channel under a pressureof 15 psi, which caused the initially large droplets sitting above thereservoir to shrink substantially (i.e., droplets 2205-2208), whiledroplets stored in the outer wells (droplets 2201-2204) shrunk muchless.

As shown in FIG. 13 C, pure water was circulated in the reservoirchannel under 15 psi pressure, which caused the initially small dropletsto swell (i.e., droplets 2205-2208) because the droplets containedsaline solution. In this fashion, all solute concentrations of thestored droplets was reversibly varied. The outer pair of droplets storedfarther away from the reservoir channels (droplets 2201-2204) changedsize much slower than the droplets stored directly above the reservoirchannels (droplets 2205-2208) and approximated the initial dropletconditions.

Although water does dissolve slightly into the bulk of the PDMSmicrofluidic device and into the carrier oil, this experimentdemonstrates that diffusion through the thin PDMS membrane is thedominant mechanism governing drop size, and not solubilization of thedroplets in the carrier oil or in the bulk of the PDMS device.

Example 4

FIG. 14 shows use of the microfluidic structure generally illustrated inFIG. 8 to perform reversible microdialysis, particularly, for thecrystallization and dissolving of the protein xylanase. The microfluidicstructure was made according to the procedures generally described inExample 1. Solutions of xylanase (4.5 mg/mL, Hampton Research), NaCl(0.5 M, Sigma-Aldrich), and buffer (Na/K phosphate 0.17 M, pH 7) wereintroduced into inlets 2050, 2055, and 2060 and were combined as aqueousco-flows. Oil was introduced into inlets 2045 and 2065. All fluids wereintroduced into the device using syringe pumps (Harvard Apparatus,PHD2000 Programmable). Droplets of the combined solution were formedwhen the solution and the oil passed through a nozzle located atintersection 2075. One hundred identical droplets, each having a volumeof 2 nL, were stored in microwells of device 2010.

Device 2010 comprised two layers. The upper layer comprised flowchannels and microwells which contained the droplets of protein. Thelower layer comprised five independent dialysis reservoirs and valvesthat controlled flow in the protein-containing channels of the upperlayer. The two layers were separated by a 15 μm thick semi-permeablebarrier 2150 made in PDMS. Square posts 2145 of PDMS covered 25% of thereservoir support the barrier. FIG. 15B is a photograph of device 2010showing microwells 2130 and square posts 2145 that supported barrier2150.

Crystallization occurred when dry air was introduced into the reservoir(i.e., at a pressure of 15 psi), which caused water to flow from theprotein solution across the barrier and into the reservoir. Oncenucleated, the crystals grew to their final size in under 10 seconds.Over 90% of the wells were observed to contain crystals. Next, air inthe reservoir was replaced with distilled water (i.e., pressurized at 15psi). Diffusion of water into the droplet caused the volume of motherliquor surrounding the crystals to increase immediately (FIG. 15C).After 15 minutes, the crystals began to dissolve rapidly and disappearedin another minute. These experiments demonstrate the feasibility ofusing a microfluidic device of the present invention to crystallizeproteins using nanoliter volumes of sample, and the ability of thesedevices to perform reversible dialysis.

Example 5

FIG. 16A is a diagram showing the energy required for nucleating acrystal. Specifically, FIG. 16A relates free energy of a sphericalcrystal nucleus (ΔG) to the size of the crystal nucleus (r). Nucleationis an activated process because a crystal of small size costs energy toform due to the liquid-crystal surface energy (γ). The free energy of aspherical crystal nucleus of radius r is ΔG=γ4πr²−Δμ4πr³/3. The heightof the nucleation barrier (ΔG*) and critical nucleus (r*) decrease asthe chemical potential difference (Δμ) between the crystal and liquidphases increases. A highly supersaturated solution (i.e., large Δμ) willhave a high nucleation rate, Γ˜exp(−ΔG*/kT) and crystals, oncenucleated, will grow rapidly.

Example 6

The following example is a prophetic example. FIG. 17 is a schematicdiagram of a typical protein phase diagram showing the relationshipbetween precipitation concentration and protein concentration in adroplet. Experiments will be performed in the device of FIG. 15.Initially, sets of droplets in wells over each of the five reservoirs(e.g., reservoirs 2140-1, 2140-2, 2140-3, 2140-4, and 2140-5 of FIG.15A) will contain protein solutions of different compositions(triangles). The reservoirs' precipitant concentrations are indicated ashorizontal dashed lines. Each protein solution (triangles) canequilibrate with its associated reservoir through the exchange of waterbetween the reservoir and protein solutions. The state of the five setsof protein solutions after equilibration are shown as follows: Solutionsremain soluble (open circles); solutions enter two-phase region (filledcircles) and phase separate into crystals; and entire solution becomescrystalline (squares). This experiment will demonstrate that entirephase diagrams can be obtained using a single microfluidic device of thepresent invention.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of or “exactly one of,” or, when used inthe claims, “consisting of,” will refer to the inclusion of exactly oneelement of a number or list of elements. In general, the term “or” asused herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of, when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of and “consistingessentially of shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is: 1-25. (canceled)
 26. A microfluidic chip,comprising: a network adapted for a directional fluid flow along a fluidpath that comprises a plurality of sections in a serial arrangement,each section comprising: a first channel that receives the directionalfluid flow; a junction fluidicly coupled to the first channel; a secondchannel fluidicly coupled to the junction and to the first channel of asubsequent section; and a microwell fluidicly coupled to the junctionand comprising a constricted fluid path that exits the microwell;wherein the directional fluid flow follows a path of lower hydrodynamicresistance through the microwell when the constricted fluid path isopen, and through the second fluid channel when the constricted fluidpath is substantially plugged.
 27. The microfluidic chip of claim 26,wherein: greater than 50% of the directional fluid flow follows a pathof lower hydrodynamic resistance through the microwell when theconstricted fluid path is open.
 28. The microfluidic chip of claim 26,wherein: greater than 60% of the directional fluid flow follows a pathof lower hydrodynamic resistance through the microwell when theconstricted fluid path is open.
 29. The microfluidic chip of claim 26,wherein: greater than 70% of the directional fluid flow follows a pathof lower hydrodynamic resistance through the microwell when theconstricted fluid path is open.
 30. The microfluidic chip of claim 26,wherein: greater than 80% of the directional fluid flow follows a pathof lower hydrodynamic resistance through the microwell when theconstricted fluid path is open.
 31. The microfluidic chip of claim 26,wherein: greater than 90% of the directional fluid flow follows a pathof lower hydrodynamic resistance through the microwell when theconstricted fluid path is open.
 32. The microfluidic chip of claim 26,wherein: the network comprises 5 or more sections.
 33. The microfluidicchip of claim 26, wherein: the network comprises 10 or more sections.34. The microfluidic chip of claim 26, wherein: the network comprises 30or more sections.
 35. The microfluidic chip of claim 26, wherein: thenetwork comprises 70 or more sections.
 36. The microfluidic chip ofclaim 26, wherein: the network comprises 100 or more sections.
 37. Themicrofluidic chip of claim 26, wherein: the network comprises 200 ormore sections.
 38. The microfluidic chip of claim 26, wherein: thenetwork comprises 500 or more sections.
 39. The microfluidic chip ofclaim 26, wherein: the network comprises 1000 or more sections.
 40. Themicrofluidic chip of claim 26, wherein: the junction further comprises athird channel fluidicly coupled to the junction.
 41. The method of claim26, wherein: the constricted fluid path is substantially plugged by anelement positioned in the microwell.
 42. The method of claim 41,wherein: the element comprises an aqueous droplet.
 43. The method ofclaim 26, wherein: the constricted fluid path is substantially pluggedby a plurality of elements positioned in the microwell.
 44. The methodof claim 41, wherein: the directional fluid flow comprises a pluralityof the elements in a serial arrangement, wherein the elements follow thepath of lower hydrodynamic resistance until positioned in the microwellof a section.
 45. The method of claim 26, wherein: the network comprisesan inlet coupled to a channel fluidicly coupled to the junction of afirst section.
 46. The method of claim 26, wherein: the directionalfluid flow comprises a flow of a carrier fluid.
 47. The microfluidicchip of claim 26, wherein: the junction further comprises a valveadapted to restrict the directional fluid flow through the microwell.48. The microfluidic chip of claim 47, wherein: the valve comprises anopen position and a closed position, wherein the directional fluid flowfollows a path of lower hydrodynamic resistance through the microwellwhen the valve is in the open position, and through the second fluidchannel when the valve is in the closed position.